Antimicrobial peptides and methods of using the same

ABSTRACT

Aspects of the present invention relate to peptides having antimicrobial activity. In certain aspects, the invention relates to peptides having potent antimicrobial activity, broad-spectrum antimicrobial activity, and/or the ability to kill otherwise antibiotic-resistant microbes, or microbes protected by biofilms.

GOVERNMENT RIGHTS

This invention was made with Government support under contract1R434EY024463-01 awarded by the National Institutes of Health andcontract W81XWH-15-1-0616 awarded by the Department of Defense. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The technology disclosed and claimed below relates generally to peptideshaving antimicrobial activity. More particularly, the invention relatesto peptides having potent antimicrobial activity, broad spectrumanti-bacterial activity, and/or the ability to kill otherwiseantibiotic-resistant bacteria, or bacteria protected by biofilms.

INTRODUCTION

Antibiotic resistance is a major health problem. This is attributed inpart to the widespread use of antibiotics not only in medicine, but alsoin agriculture and animal husbandry. Such overuse of antibiotics, whilekilling susceptible organisms, has also created a powerful selectionbias toward antibiotic resistant bacteria. The resulting antibioticresistant strains pose a particular problem for individuals withweakened immune systems and represent an increasingly serious problemfor patients in hospitals. Acute bacterial skin and skin structureinfections (ABSSSI) are responsible for 730,000 hospitalizations peryear at substantial cost. Broad-spectrum coverage for gram-negativepathogens and multidrug-resistant gram-positive bacteria, such ascommunity-acquired methicillin resistant Staphylococcus aureus (MRSA)strains are limited and frequent outbreaks occur.

In addition to exhibiting inherited antibiotic resistance, many emergingbacterial strains can exist in complex associations known as biofilms,densely packed communities of microbial cells that can grow on living orinert surfaces and surround themselves with secreted polymers. Thestructure of a biofilm constitutes a physical barrier to antibioticexposure. Biofilms are 20-1000 times more resistant to antibiotics thantheir planktonic counterparts and can form in and on tissues,particularly on chronic wounds and medical implants, such as indwellingcatheters, artificial organs, and the like, where they have thepotential to cause systemic infections requiring heroic treatments.There is an urgent need for materials that are active against antibioticresistant organisms in both free and biofilm form.

As part of their natural defense against bacteria, many organismsincluding insects, amphibians, mammals, and humans, produceantimicrobial peptides. Such peptides are chemically diverse. Someantimicrobial peptides appear to act by penetrating the bacterial cellmembrane and destroying it. Other antimicrobial peptides affectbacterial cellular processes. Considerable selectivity is observed, withmany of the peptides targeting bacteria in preference to host cells.Unfortunately, host-produced antimicrobial peptides are generally notcapable of effectively eliminating a wide range of microbial agents,including many antibiotic resistant bacterial strains. Antimicrobialpeptides having potent antimicrobial activity, broad spectrumanti-bacterial activity, and/or the ability to kill otherwiseantibiotic-resistant bacteria are of interest.

SUMMARY

The invention provides antimicrobial peptides and methods of using thesame. The peptides of this disclosure can have anti-bacterial,anti-fungal, and/or anti-protozoal activity. The peptides can killmicrobial strains that are resistant to conventional antibiotics. Thisdisclosure provides antimicrobial peptides that are capable of killingmicrobes (e.g., bacteria) growing as a microbial biofilm. The technologydescribed and claimed below represents the first description ofparticular types of antimicrobial peptides that can be used a variety ofapplications, e.g., to selectively kill microbes (e.g., bacteria) forpurposes of treatment of conditions related to microbial infections.

The invention is put forth in the description that follows, in thefigures, and in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary bubble region-containing peptide of thedisclosure. The peptide depicted includes a bubble region having adisulfide bond that is flanked by amphipathic regions 1 and 2.

FIG. 2A and FIG. 2B depict peptides including a cysteine-containingbubble region flanked by two amphipathic regions that themselves includeadditional cysteine residues. FIG. 2A depicts a monomeric version of thepeptide where cysteine residues located in the amphipathic regions maybe linked via an intramolecular disulfide bond. FIG. 2B depicts adimeric version of the peptide where the two cysteine residues may belinked via an intermolecular disulfide bond.

FIG. 3A to 3C shows RP557 rapidly eradicates P. aeruginosa and S. aureuswith no cytotoxicity to mammalian cells. Cell viability was performedusing bioluminescent strains of P. aeruginosa 19660 (FIG. 3A), S. aureus49525 (FIG. 3B) & L929 fibroblast cells (FIG. 3C).

FIG. 4A to 4B shows data indicating that P. aeruginosa and S. aureus didnot develop resistance against RP557. Sub-inhibitory concentrations ofRP557, gentamicin and clindamycin were incubated with P. aeruginosa27853 and S. aureus 29213 for 24 hours. Bacteria showing growth in thehighest concentration were re-passaged in fresh dilutions containingsub-minimum inhibitory concentration (MIC) levels of each component for30 consecutive passages.

FIG. 5A to 5B show data indicating RP557 is a potent inhibitor ofCandida biofilm. Fluconazole (FIG. 5A) or RP557 (FIG. 5B) were added topreformed Candida 17-88 biofilm for 24 hours and biofilm inhibitionevaluated via metabolic evaluation using XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl) [phenyl-amino)carbonyl]-2H.

FIG. 6A to 6B shows that topical treatment with exemplary peptide RP557reduces polymicrobial infection in an infected porcine burn model. Fullthickness wounds were created with a heated brass rod and a trephine onthe back of anesthetized pigs followed by infection with 2:2:1 mixtureof S. aureus ATCC 6538 and P. aeruginosa (porcine isolate) andfusobacterium ssp.

FIG. 7A to 7B show that exemplary peptide RP557 kills infectionthroughout a 24 hour period. Pharmacodynamic Response to RP557. 24 hourspost bacterial inoculation biofilm associated wounds were treated with2% RP557. Punch biopsies were taken at 30 min, 180 min and 24 hours posttreatment, homogenized, plated and CFUs counted.

FIG. 8A to 8B indicates topical treatment of exemplary peptide RP557reduces rodent vaginal candidiasis. Effects of RP557, miconazole andoral fluconazole in a C. albicans vaginal infection rodent model. On Day0, rats were inoculated intravaginally with C. albicans. RP557 andmiconazole were administered twice daily (FIG. 8A). A separate groupreceived oral fluconazole (FIG. 8B). Further details are provided in theexperimental section.

DETAILED DESCRIPTION

As discussed above, the invention disclosed herein relates toantimicrobial peptides and methods of administering such antimicrobialpeptides to a subject to prevent or treat a microbial infection.

Antimicrobial Peptides

Certain features of the antimicrobial peptides of this disclosure willnow be described in more detail. The antimicrobial peptides generallyinclude an amphipathic or striapathic region. In some cases, the peptideincludes one contiguous amphipathic or striapathic region. In somecases, the peptide includes two amphipathic or striapathic regionsseparated by a linking region or bubble region (e.g., as describedherein).

By “amphipathic region” is meant a peptide region or segment thatpossesses both hydrophobic and hydrophilic structural elements orcharacteristics, for example, a peptide region capable of possessing astructure having a hydrophilic surface and a hydrophobic surface. Apeptide region is said to be amphipathic and in an amphipathicconformation when it exhibits an amphipathic characteristic. Theamphipathic characteristics of a peptide can be dependent in part on theenvironmental conditions to which the peptide is subjected and/orutilized, e.g., aqueous conditions or physiological conditions. To beconsidered or referred to as amphipathic, a peptide sequence (or portionthereof) need not be in an amphipathic conformation at all times.Rather, it is sufficient that in some cases the amphipathic conformationbe present, e.g., the peptide is capable of adopting an amphipathicconformation under suitable conditions, such as the conditions in whichthe peptide finds use.

An amphipathic region of a peptide can be referred to as a striapathicregion. The term “striapathic region” refers to a region or portion of apeptide sequence that is composed of a sequence of alternatinghydrophobic and hydrophilic modules. A “hydrophobic module” is a peptidesequence consisting of one to five (e.g., 1 to 3 or 1 to 2) hydrophobicamino acid residues, e.g., 1, 2, 3, 4 or 5 hydrophobic amino acidresidues. A “hydrophilic module” is a peptide sequence consisting of oneto five (e.g., 1 to 3 or 1 to 2) hydrophilic amino acid residues, e.g.,1, 2, 3, 4 or 3 hydrophilic amino acid residues.

Hydrophobic amino acid residues are characterized by a sidechain groupthat has predominantly non-polar chemical or physical properties, e.g.,in an environment in which a peptide finds use. e.g., physiologicalconditions. Such hydrophobic amino acid residues can be naturallyoccurring or non-naturally occurring. A hydrophobic amino acid residuecan be a mimetic of a naturally occurring amino acid that ischaracterized by a sidechain group that has predominantly non-polarchemical or physical properties. Conversely, hydrophilic amino acidresidues are characterized by a sidechain group that is predominantlypolar (e.g., charged or neutral hydrophilic), e.g., in an environment inwhich a peptide finds use, e.g., physiological conditions. Suchhydrophilic amino acid residues can be naturally occurring ornon-naturally occurring. A hydrophilic amino acid residue can be amimetic of a naturally occurring amino acid characterized by a sidechaingroup that is predominantly hydrophilic (charged or neutral polar).Examples of hydrophilic and hydrophobic amino acid residues are shown inTable 1, below. The hydrophobic and hydrophilic amino acid residues canbe L-amino acid residues. The hydrophobic and hydrophilic amino acidresidues can be D-amino acid residues. Suitable non-naturally occurringamino acid residues and amino acid mimetics that can find use in thesubject peptides are readily available in the art. See, e.g., Liang etal. (2013), “An Index for Characterization of Natural and Non-NaturalAmino Acids for Peptidomimetics,” PLoS ONE 8(7):e67844.

Although most amino acid residues can be considered as eitherhydrophobic (non-polar) or hydrophilic (polar or charged), a few,depending on the context, can behave as either hydrophobic orhydrophilic.

TABLE 1 Hydrophobic and Hydrophilic Amino Acid Residues HydrophilicHydrophobic Residues Residues (X) (J) lysine phenylalanine ornithinetryptophan arginine tyrosine histidine isoleucine aspartic acid leucineglutamic acid valine asparagine methionine glutamine alanine pyrrolysineproline glycine cysteine threonine norleucine norvaline

As described in further detail below, aspects of the present disclosureinclude antimicrobial peptides having at least one amphipathic orstriapathic region having a specific degree of cationic charge. Incertain embodiments, the antimicrobial peptide includes a tail region(e.g., a hydrophobic tail sequence). In certain embodiments, anantimicrobial peptide (or peptide agent) includes two or moreamphipathic or striapathic regions. In such embodiments, two amphipathicregions of an antimicrobial peptide are in the form of a dimer, wherethe two amphipathic regions can have the same or different amino acidsequences (i.e., be homodimer or a heterodimer). In certain embodiments,the two (or more) amphipathic or striapathic regions are connected via alinker or linking region. The linker can be a contiguous (or in-line)amino acid sequence or a non-amino acid moiety as desired by a user. Thelinking region can be, e.g., a bubble region or a beta-turn region.

In certain embodiments, an amphipathic region of a peptide is referredto as a striapathic region and includes an alternating sequence composedof 1 to 5 hydrophobic residues (a hydrophobic module J₁₋₅) followed by 1to 5 hydrophilic amino acid residues (a hydrophilic module X₁₋₅). Astriapathic region can thus be represented by the formulae(X₁₋₅J₁₋₅)_(n) or (J₁₋₅X₁₋₅)_(n), where each X signifies a hydrophilicamino acid residue, each J signifies a hydrophobic amino acid residue,and each n is an integer from 1 to 15, such as 2 to 15, 2 to 10, 3 to10, 4 to 10 or 5 to 10. For example, an amphipathic region can have asequence according to Formula 1, Formula 2 (the reverse of Formula 1) orFormula 3:

Formula 1: X¹J²J³X⁴X⁵J⁶J⁷X⁸X⁹J¹⁰X¹¹X¹²J¹³J¹⁴X¹⁵X¹⁶J¹⁷J¹⁸ Formula 2:J¹J²X³X⁴J⁵J⁶X⁷X⁸J⁹X¹⁰X¹¹J¹²J¹³X¹⁴X¹⁵J¹⁶J¹⁷X¹⁸ Formula 3:J¹X²J³X⁴J⁵X⁶J⁷X⁸J⁹X¹⁰J¹¹X¹²J¹³X¹⁴J¹⁵X¹⁶J¹⁷

Each hydrophobic amino acid residue J is selected from the groupconsisting of a naturally occurring hydrophobic amino acid, anon-naturally occurring hydrophobic amino acid, and a hydrophobic aminoacid mimetic. Each hydrophilic amino acid residue X is selected from thegroup consisting of a naturally occurring hydrophilic amino acid, anon-naturally occurring hydrophilic amino acid, and a hydrophilic aminoacid mimetic. Often, the amphipathic conformation will be associatedwith a particular secondary structure, such as a helical structure.Thus, the amphipathic region of an antimicrobial polypeptide can have anamphipathic 3₁₀-helical conformation, an amphipathic α-helicalconformation, an amphipathic π-helical conformation, or an amphipathicpolyproline helical conformation. Alternatively, the amphipathic regionof an antimicrobial polypeptide can have an amphipathic β-strandconformation.

In certain embodiments, the amphipathic region of an antimicrobialpeptide according to aspects of the present disclosure includes one ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) large hydrophobic aminoacid residues. Examples of large hydrophobic amino acid residues includetryptophan, phenylalanine, and tyrosine. In addition, under certaincircumstances, histidine can be considered a large hydrophobic aminoacid residue. In certain embodiments, the amphipathic region of anantimicrobial peptide according to aspects of the present disclosureincludes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) smallhydrophobic amino acid residues. Examples of small hydrophobic residuesinclude glycine, alanine, valine, leucine, threonine, and proline. Inaddition, under certain circumstances serine or cysteine can beconsidered a small hydrophobic residue. In certain embodiments, theantimicrobial polypeptide has an amphipathic region that includes acombination of large and small hydrophobic residues.

Cationic Charge/Surface

Antimicrobial polypeptides according to aspects of the presentdisclosure can include an amphipathic region having a cationic surface.In certain embodiments, the amphipathic region has a cationic charge(i.e., charge >0, e.g., +1, +2, +3, +4, +5, +6, +7, +8, +9, +10 ormore). Thus, in certain embodiments, an amphipathic region of thedisclosed peptides contains one or more polar cationic amino acidresidues (i.e., residues having positively charged side chains), such as2 or more, 3 or more, 4 or more, or 5 or more polar cationic amino acidresidues, in some cases, up to 10 polar cationic amino acid residues.Examples of amino acid residues having positively charged side groups(assuming physiological conditions) include lysine, ornithine andarginine, and sometimes histidine. Accordingly, an antimicrobialpolypeptide can have an amphipathic region that includes from 1 to 20cationic amino acid residues, such as 2 to 20, 3 to 30, 4 to 20, 5 to20, 6 to 20, 8 to 20 or 10 to 20 cationic amino acid residues. Thus, anantimicrobial peptide of the invention can include polar amino acidresidues, at least 40% (e.g., 50%, 60%, 70%, 80%, 90%, or 100%) of whichare cationically charged (e.g., are selected from Arg, Lys and Orn).

Tail Region

In certain embodiments, an antimicrobial peptide includes a tail region.A tail region of an antimicrobial peptide of the invention can be from 3to 15 amino acid residues in length, with at least 50% (e.g., at least60%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or more) of the amino acid residues in the tail region beinghydrophobic. The tail region can be located at cither the N-terminus,the C-terminus, or both termini of the antimicrobial peptide. Thisdisclosure provides peptides having a tail region including a sequenceof 3, 4 or 5 hydrophobic amino acids. This disclosure provides peptideshaving a tail region including a sequence of 3-6 amino acids having oneor lacking a hydrophilic amino acid residue. In certain embodiments, thetail region includes one hydrophilic amino acid for every 6 amino acidsin the sequence. In some cases, a tail region sequence is shown inFormula 4, where each J signifies a hydrophobic amino acid residue(e.g., of Table 1):

Formula 4: J¹J²J³J⁴J⁵.

In some embodiments of Formula 4, J¹, J³ and J⁵ are independently alarge hydrophobic residue (e.g., tryptophan, phenylalanine, andtyrosine), and J² and J⁴ are independently a small hydrophobic residue(e.g., glycine, alanine, valine, leucine, threonine, and proline). Insome embodiments of Formula 4, each J is independently a hydrophobicamino acid residue selected from Phe and Ala. In some cases of Formula4, the tail region includes the sequence FAFAF (SEQ ID NO: 19). In someinstances of Formula 4, the tail region includes the sequence AFAFA (SEQID NO: 20).

Specific examples of antimicrobial peptides according to aspects of thisdisclosure that include a tail region having the sequence FAFAF (SEQ IDNO: 19) include the following where the tail region is underlined:

(RP-551, SEQ ID NO: 2) FIOKFAKOFKOFIOKFAKFAFAF; and(RP-552, SEQ ID NO: 3) FAFAFKAFKKAFKOFOOAFOOAF.Bubble Region

In certain embodiments, an antimicrobial peptide includes a bubbleregion. A “bubble” region of an antimicrobial peptide of this disclosureconsists of a stretch of amino acid residues flanked by cross-linkingresidues (e.g., cysteine residues (C) capable of disulfide formation) ator near each end of the region (see FIG. 1). The sequence of amino acidresidues located between the flanking cross-linking residues (e.g.,cysteine residues) can be from 2 to 10 amino acid residues in length. Incertain cases, the stretch of amino acid residues between thecross-linking residues (e.g., cysteine residues) is from 3 to 6 aminoacid residues in length, such as 3 to 5 amino acid residues in length,or in particular 3, 4 or 5 amino acid residues. A bubble region can linktwo amphipathic regions and contribute to the formation of a desirablehairpin secondary structure by the antimicrobial peptide. In someinstances, the bubble region includes an intramolecular crosslink. Suchan intramolecular crosslink, if present, can be covalent (e.g., adisulfide) or non-covalent (e.g., a salt bridge). In some cases, thebubble region includes a disulfide bond between two flanking cysteineresidues (see FIG. 1). This region can thus be classified as a type of“linker region” (as can other regions, described elsewhere herein). Incertain embodiments, the hairpin secondary structure can significantlyenhance antimicrobial activity.

A bubble region can include a two cysteine residues linked via asequence of any 4 amino acids, e.g., a sequence C(AA¹)AA²)(AA³)(AA⁴)C.Optionally, the cysteine residues of the bubble region may be linked tothe adjacent stripathic regions of the peptide by a linker, such as oneor more linking residues. In some cases, the one or more linkingresidues are glycine residue(s), e.g., a sequence(G)_(n)C(AA¹)AA²)(AA³)(AA⁴)C(G)_(m) where n and m are independently 0-6,such as 0, 1, 2, 3 or 4.

A bubble region can include, for example, a sequence as shown in Formula5, where each J signifies a hydrophobic amino acid residue and each Xsignifies a hydrophilic amino acid residue:

Formula 5: C(J/X)(J/X)(J/X)(J/X)C.

In some instances of Formula 5, the bubble region has the sequence:

Formula 5b: CJ(X/J)(X/J)JC

In some instances of Formula 5b, the bubble region has one of thefollowing sequences:

Formula 5c: CJGXJC Formula 5d: CJXGJC Formula 5e: CJGGJC.

In some instances of Formula 5c-5e, each J is selected from F, L, I, Vand A. In some instances of Formula 5c-5d, X is selected from K, R andO.

Specific examples of antimicrobial peptides of this disclosure thatinclude a bubble region having the sequence CLGRFC (SEQ ID NO: 21) orCYKGIC (SEQ ID NO: 22) include (the bubble region is underlined):

(RP-550, SEQ ID NO: 1) FKIOARLCLGOFCIOARLK; and (RP-557, SEQ ID NO: 8)RFCWKVCYKGICFKKCK.

In some embodiments antimicrobial peptides of the invention include abubble region, and one or more additional linked regions. In certainembodiments, antimicrobial peptides of the invention that include abubble region also include one or more additional stretches of aminoacid residues flanked by cysteine residues positioned on either side ofthe bubble region. For example, a subject antimicrobial peptide mayinclude four cysteine residues, with two cysteine residues forming abubble region and the remaining two cysteine residues being positionedon opposing sides of the two amphipathic regions either side of thebubble region so as to form an additional linked region across thesecondary hairpin structure (see FIG. 1B). Specific examples ofantimicrobial peptides of the invention that include a bubble regionhaving the sequence CYKGIC (SEQ ID NO: 22) and include an additionallinked region include (the bubble region and additional linked region isunderlined):

(RP-556, SEQ ID NO: 7) RWCFKVCYKGICYKKCK (RP-557, SEQ ID NO: 8)RFCWKVCYKGICFKKCKDimerization

Without intending to be limited by theory, in some cases, efficacy ofthe antimicrobial peptides of the invention depends, in part, on peptidedimerization and clustering on the cell membrane of the target microbe(e.g., bacterial cell). It is believed that dimers can be efficient atpenetrating and, ultimately, lysing the cell membrane. The formation ofsuch dimers can be thermodynamically more favorable when the peptidesare physically linked together, e.g., using linker regions orcross-linking amino acid residues. See FIG. 2B. Linker regions caninclude additional amino acid residues (e.g., like the bubble regiondescribed above) or be non-amino acid-containing linker moieties.

Beta Turn Region

A β-turn sequence can be used to physically link two regions, makingintra-molecular interactions more likely to take place. This appears tobe particularly important for amphipathic lytic peptides, as it allowstheir hydrophobic surfaces to be protected from the aqueous phase. Abubble region (e.g., as described here) can provide for a similarconfiguration. The β-turn sequence allows for two intra-chainamphipathic regions to form a dimer in an antiparallel orientation. Thisregion can thus be classified as a type of “linker region” (as can otherregions, described elsewhere herein).

A β-turn sequence that finds use in linking two striapathic regions canbe any β-turn sequence known in the art. A β-turn sequence can have, forexample, a sequence as shown in SEQ ID NO: 23, where J signifieshydrophobic amino acid residues and X signifies hydrophilic amino acidresidues (i.e., any amino acid residue).

(SEQ ID NO: 23) (J/X)GPGR(J/X)

Exemplary antimicrobial peptide sequences are described below thatinclude one or more of the features described above. Additional peptidesof this disclosure can be readily designed by one skilled in the art bycombining different regions or features of the exemplary antimicrobialpeptides in different ways as described herein.

Examples of Antimicrobial Peptides

Particular antimicrobial peptides of interest, and fragments andvariants thereof which find use in the subject pharmaceuticalcompositions and methods are now described in greater detail. In certaincases, the subject antimicrobial peptides are of 10 to 50 amino acidresidues in length, such as 10 to 40, 10 to 30, 10 to 20, or 12 to 30,13 to 30, 14 to 30, 15 to 30, 15 to 25, 15 to 20, 15 to 19, or 17 to 30,17 to 25, or 17 to 20 amino acid residues in length. The subject peptidecan comprise a striapathic region of alternating hydrophilic andhydrophobic modules that adopts an amphipathic conformation underphysiological conditions (e.g., as described herein). In certaininstances, the striapathic region of a peptide is of 5 to 30 amino acidresidues in length, such as 5 to 25, 5 to 20, 5 to 19, 6 to 18, 6 to 17,7 to 18 amino acid residues in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length). The striapathicregion can comprise: 2 or more (e.g., 3 or more, 4 or more, 5 or more,or 6 or more) hydrophobic modules; and one or more (e.g., 3 or more, 4or more, 5 or more, or 6 or more) hydrophilic modules. In some cases,each hydrophilic module includes at least one cationic residue. In someinstances, the peptide has one and only one striapathic region of 10 to30 amino acid residues in length, such as 12 to 30, 15 to 25 or 10 to 20or 15 to 20 amino acid residues in length (e.g., 15, 16, 17, 18, 19 or20 amino acid residues in length). In some instances, the peptide hastwo striapathic regions linked via a bubble region or linking region,where each striapathic region is independently 5 to 15 amino acidresidues in length, such as 5 to 12, 5 to 10, or 6 to 12 or 6 to 10amino acid residues in length (e.g., 5, 6, 7, 8, 9 or 10 amino acidresidues in length).

In certain instances, a striapathic region of the peptide is of 5 to 30amino acid residues in length (e.g., 6 to 20, 6 to 19, 7 to 19, 14, 15,16, 17, 18 or 19 amino acids in length), wherein the peptide isoptionally further modified (e.g., as described herein). The striapathicregion can comprise: 2 or more (e.g., 3 or more or 4 or more)hydrophobic modules; and one or more (e.g., 2 or more or 3 or more)hydrophilic modules each comprising at least one cationic residue. Insome instances, the striapathic region of the peptide has a length of 7to 20 amino acid residues.

The hydrophobic modules can consist of any convenient residues. Incertain instances, the hydrophobic modules include amino acid residuesselected from phenylalanine, tryptophan, alanine, valine, glycine,isoleucine, leucine, cysteine and tyrosine. The striapathic region caninclude 2 or more cationic amino acid residues in total, such as 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,or even more. The antimicrobial peptide can include a striapathic regioncomprising 3 or more hydrophilic modules that consist of any convenienthydrophilic residues. In some instances, the hydrophilic modules includeamino acid residues selected from lysine, arginine, histidine, asparticacid, glutamic acid, asparagine, glutamine and ornithine. In certaininstances, the hydrophilic modules include amino residues selected fromlysine, arginine and ornithine.

In certain embodiments, the antimicrobial peptide comprises three ormore hydrophilic modules (such as 3, 4, 5 or 6, or more) and three ormore hydrophobic modules (such as 3, 4, 5 or 6, or more) and is apeptide of Formulae 1 or 2. Formulae 1 and 2 can be represented asfollows to show the hydrophilic and hydrophobic modules:

Formula 1: [X¹]-[J²J³]-[X⁴X⁵]-[J⁶J⁷]-[X⁸X⁹]-[J¹⁰]-[X¹¹X¹²]-[J¹³J¹⁴]-[X¹⁵X¹⁶]-[J¹⁷J¹⁸] Formula 2:[J¹J²]-[X³X⁴]-[J⁵J⁶]-[X⁷X⁸]-[J⁹]-[X¹⁰X¹¹]-[J¹²J¹³]-[X¹⁴X¹⁵]-[J¹⁶J¹⁷]-[X¹⁸]wherein, each X is independently a hydrophilic amino acid residue (e.g.lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine,glutamine or ornithine); and each J is independently a hydrophobicresidue (e.g. phenylalanine, tryptophan, alanine, valine, glycine,isoleucine, leucine, or tyrosine). It is understood that, in some cases,an antimicrobial peptide of Formula 1 has a sequence which can bereversed to provide an antimicrobial peptide having a sequence ofFormula 2.

In some cases of formula 1, each X is independently selected fromarginine, lysine and ornithine. In some cases of formula 1, each X isindependently selected from lysine and ornithine. In some cases offormula 1, each J is independently selected from phenylalanine, alanine,isoleucine, leucine and valine. In some cases of formula 1, each J isindependently selected from phenylalanine and alanine. In some cases offormula 2, each X is independently selected from arginine, lysine andornithine. In certain cases of formula 2, each X is independentlyselected from lysine and ornithine. In some cases of formula 2, each Jis independently selected from phenylalanine, alanine, isoleucine,leucine and valine. In some cases of formula 2, each J is independentlyselected from phenylalanine, alanine and isoleucine. In some cases theantimicrobial polypeptide of formulae 1 or 2 includes one or moreadditional hydrophilic or hydrophobic residues at the C terminal or theN terminal. In some cases, the antimicrobial polypeptide of formulae 1or 2 includes a tail region (e.g., as described herein) at the Cterminal or the N terminal.

Formula 3 Peptides

In some embodiments, the antimicrobial polypeptide comprises a sequencedefined by the formula 3 in which each residue in the sequencealternates between a hydrophobic residue (J) and a hydrophilic residue(X):

Formula 3: J¹X²J³X⁴J⁵X⁶J⁷X⁸J⁹X¹⁰J¹¹X¹²J¹³X¹⁴J¹⁵X¹⁶J¹⁷wherein, each X is independently a hydrophilic amino acid residue; andeach J is independently a hydrophobic residue.

In some cases of formula 3, each X is independently selected fromlysine, arginine, histidine, aspartic acid, glutamic acid, asparagine,glutamine and ornithine. In some cases of formula 3, each X isindependently selected from ornithine, lysine and arginine. In somecases of formula 3, each X is independently selected from ornithine andlysine. In some cases of formula 3, each J is independently selectedfrom phenylalanine, tryptophan, alanine, valine, glycine, isoleucine,leucine and tyrosine. In some cases of formula 3, each J isindependently selected from phenylalanine, alanine, isoleucine, leucineand valine. In certain cases of formula 3, the antimicrobial polypeptideincludes one or more additional hydrophilic or hydrophobic residues atthe C terminal and/or the N terminal. In some cases of formula 3, theantimicrobial peptide includes a tail region (e.g. as described herein)at the C terminal and/or the N terminal.

In some embodiments of the antimicrobial polypeptide of formula 3, thesequence has a formula defined by the formula 3A:

Formula 3A: FX²J³X⁴J⁵X⁶J⁷X⁸J⁹X¹⁰J¹¹X¹²J¹³X¹⁴J¹⁵X¹⁶J¹⁷wherein:

X² and X¹⁴ are each independently selected from O and R;

J³ and J¹⁷ are each independently selected from L and I;

X⁴ and X¹⁶ are each independently selected from K and O;

J⁵ is selected from A and I;

X⁶, X⁸, X¹⁰ and X¹² are each independently selected from R, K and O;

J⁷ is selected from F, A and I;

J⁹ is selected from V and L;

J¹¹ is selected from A, V and L;

J¹³ is selected from A, I and L; and

J¹⁵ is selected from I, F and L.

In some instances of formula 3A, the antimicrobial peptide includes apeptide sequence FRLKIKARLKVKIRFKL (RP554) (SEQ ID NO: 5), or a variantor fragment thereof.

In some embodiments of the antimicrobial polypeptide of formula 3, thesequence has a formula defined by the formula 3B:

Formula 3B: (SEQ ID NO: 24) FOJ³X⁴AX⁶J⁷X⁸VX¹⁰J¹¹X¹²J¹³OJ¹⁵X¹⁶J¹⁷wherein:

J³, J¹⁵ and J¹⁷ are each independently selected from I and L;

X⁴, X⁸ and X¹⁶ are each independently selected from O and K;

X⁶ and X¹² are each independently selected from O and R;

J⁷ is selected from I and F;

X¹⁰ are each independently selected from O, K and R; and

J¹¹ and J¹³ are each independently selected from A and L.

In some instances of formula 3A or 3B, the antimicrobial peptideincludes a peptide sequence FOIKARFOVRARLOLKI (RP553) (SEQ ID NO: 4), ora variant or fragment thereof.

In some instances of formula 3A or 3B, the antimicrobial peptideincludes a peptide sequence FOLOAOIOVOLOAOIOL (RP555) (SEQ ID NO: 6), ora variant or fragment thereof.

In some instances of formula 3A or 3B, the antimicrobial peptideincludes a peptide sequence FOLOAOIKVKLOAOIOL (RP556) (SEQ ID NO: 7), ora variant or fragment thereof.

Formula 6 Peptides

The antimicrobial polypeptide can include two striapathic regions linkedvia a bubble region (e.g., as described herein).

In some embodiments, the antimicrobial polypeptide comprises a sequencedefined by the formula 6, in which two striapathic regions composed ofalternating hydrophobic residues (J) and hydrophilic residues (X) arejoined by a central bubble region (B):

Formula 6: J¹X²J³X⁴J⁵X⁶J⁷(X⁸)_(m)-B-(X¹³)_(m)J¹⁴X¹⁵J¹⁶X¹⁷J¹⁸X¹⁹J²⁰

wherein:

-   -   B is a sequence selected from the following formulae C-Z-C and        GGC-Z-CGG (SEQ ID NO: 25), wherein each C is a cysteine residue,        each G is a glycine residue and Z consists of 3-5 amino acid        residues selected from hydrophobic amino acid residues (J) and        hydrophilic amino acid residue (X) (e.g., as described herein);        and    -   m is 0 or 1.

In some embodiments of the antimicrobial polypeptide of formula 6. B isa sequence selected from the following formulae CJ⁹J¹⁰X¹¹J¹²C and(G)_(n)CJ⁹J¹⁰X¹¹J¹²C(G)_(n) (SEQ ID NO: 26), wherein each C is acysteine residue, each G is a glycine residue, each n is independentlyselected from 1-4 (e.g., 1, 2 or 3), each X is independently selectedfrom a hydrophilic amino acid residue (e.g. lysine, arginine, histidine,aspartic acid, glutamic acid, asparagine, glutamine and ornithine) andeach J is independently selected from a hydrophobic residue (e.g.phenylalanine, tryptophan, alanine, valine, glycine, isoleucine,leucine, and tyrosine).

In some cases of the peptide, J⁹, J¹⁰ and J¹² are independently selectedfrom leucine, glycine and phenylalanine. In certain cases, X¹¹ isselected from ornithine and lysine.

In some embodiments of the antimicrobial polypeptide of formula 6, thepolypeptide comprises a sequence defined by formula 6A:

Formula 6A: (SEQ ID NO: 27) FX²IX⁴AX⁶L(X⁸)_(m)-B-(X¹³)_(m)IX¹⁵AX¹⁷LX¹⁹F

wherein:

-   -   B is a sequence selected from the following formulae CLGX¹¹FC        (SEQ ID NO: 28), GCLGX¹¹FCG (SEQ ID NO: 29) and GGCLGX¹¹FCGG        (SEQ ID NO: 30), wherein each C is a cysteine residue, each G is        a glycine residue and X¹¹ is selected from O and K;    -   X², X⁴, each X⁸, each X¹³, X¹⁵ and X¹⁹ are independently        selected from O and K;    -   X⁶ and X¹⁷ are each independently selected from R and O; and    -   each m is independently an integer selected from 0 and 1.

In some instances of formula 6A, the antimicrobial peptide includes apeptide sequence FOIOAOLGGCLGOFCGGIOAOLOF (RP564) (SEQ ID NO: 15), or avariant or fragment thereof.

In some instances of formula 6A, the antimicrobial peptide includes apeptide sequence FOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO: 16), ora variant or fragment thereof.

In some instances of formula 6A, the antimicrobial peptide includes apeptide sequence FOIKAOLGGCLGKFCGGIKAOLKF (RP566) (SEQ ID NO: 17), or avariant or fragment thereof.

In some instances of formula 6A, the antimicrobial peptide includes apeptide sequence

FOIKAOLKGGCLGKFCGGKIKAOLKF (RP 567) (SEQ ID NO: 18), or a variant orfragment thereof.

In some embodiments, the antimicrobial polypeptide comprises a sequencedefined by the formula 6B, based on the formula 6:

Formula 6B: J¹X²J³X⁴J⁵X⁶J⁷-CJ⁹J¹⁰X¹¹J¹²C-J¹⁴X¹⁵J¹⁶X¹⁷J¹⁸X¹⁹wherein each C is a cysteine residue, each X is independently selectedfrom a hydrophilic amino acid residue (e.g. lysine, arginine, histidine,aspartic acid, glutamic acid, asparagine, glutamine and ornithine); andeach J is independently selected from a hydrophobic residue (e.g.phenylalanine, tryptophan, alanine, valine, glycine, isoleucine,leucine, and tyrosine). In some cases, each X is independently selectedfrom ornithine, lysine and arginine. In some cases, each J isindependently selected from phenylalanine, alanine, isoleucine, leucineand glycine.

In some instances of formula 6B, the antimicrobial peptide includes apeptide sequence FKIOARLCLGOFCIOARLK (RP550) (SEQ ID NO: 1), or avariant or fragment thereof.

In some embodiments, the antimicrobial polypeptide comprises a sequencedefined by the formula 6C, based on the formula 6:

Formula 6C: (SEQ ID NO: 31)J¹X²J³X⁴J⁵X⁶J⁷-GGCJ⁹J¹⁰X¹¹J¹²CGG-J¹⁴X¹⁵J¹⁶X¹⁷J¹⁸X¹⁹J²⁰wherein C is a cysteine residue. G is a glycine residue, each X isindependently a hydrophilic amino acid residue (e.g. lysine, arginine,histidine, aspartic acid, glutamic acid, asparagine, glutamine orornithine); and each J is independently a hydrophobic residue (e.g.phenylalanine, tryptophan, alanine, valine, glycine, isoleucine,leucine, or tyrosine). In some cases, each X is independently selectedfrom ornithine, lysine and arginine. In some cases, each J isindependently selected from phenylalanine, alanine, isoleucine, leucineand glycine.

In some instances of formula 6C, the antimicrobial peptide includes apeptide sequence FOIOAOLGGCLGOFCGGIOAOLOF (RP564) (SEQ ID NO: 15), or avariant or fragment thereof.

In some instances of formula 6C, the antimicrobial peptide includes apeptide sequence FOIKAOLGGCLGKFCGGIKAOLKF (RP566) (SEQ ID NO: 17), or avariant or fragment thereof.

In some embodiments, the antimicrobial polypeptide comprises a sequencedefined by the formula 6D, based on the formula 6:

Formula 6D: (SEQ ID NO: 32)J¹X²J³X⁴J⁵X⁶J⁷X⁸-GGCJ⁹J¹⁰X¹¹J¹²CGG-X¹³J¹⁴X¹⁵J¹⁶X¹⁷J¹⁸X¹⁹J²⁰wherein C is a cysteine residue, G is a glycine residue, each X isindependently a hydrophilic amino acid residue (e.g. lysine, arginine,histidine, aspartic acid, glutamic acid, asparagine, glutamine orornithine); and each J is independently a hydrophobic residue (e.g.phenylalanine, tryptophan, alanine, valine, glycine, isoleucine,leucine, or tyrosine). In some cases, each X is independently selectedfrom ornithine, lysine and arginine. In some cases, each J isindependently selected from phenylalanine, alanine, isoleucine, leucineand glycine.

In certain cases of the antimicrobial polypeptide of any one of formulae6B to 6D. J¹X²J³X⁴J⁵X⁶J⁷ is FX²IX⁴AX⁶L (SEQ ID NO: 33), wherein X² andX⁴ are each independently selected from O and K and X⁶ is selected fromR and O. In certain embodiments of any one of formulae 6B to 6D,FX²IX⁴AX⁶L (SEQ ID NO: 33) is selected from FKIOARL (SEQ ID NO: 34),FOIOAOL (SEQ ID NO: 35) and FOIKAOL (SEQ ID NO: 36). In some cases ofany one of formulae 6B to 6D, FX²IX⁴AX⁶L (SEQ ID NO: 33) is FKIOARL (SEQID NO: 34). In some cases of any one of formulae 6B to 6D FX²IX⁴AX⁶L(SEQ ID NO: 33) is FOIOAOL (SEQ ID NO: 35). In other cases of any one offormulae 6B to 6D, FX²IX⁴AX⁶L (SEQ ID NO: 33) is FOIKAOL (SEQ ID NO:36).

In some instances of formula 6D, the antimicrobial peptide includes apeptide sequence FOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO: 16), ora variant or fragment thereof.

In some instances of formula 6D, the antimicrobial peptide includes apeptide sequence FOIKAOLKGGCLGKFCGGKIKAOLKF (RP567) (SEQ ID NO: 18), ora variant or fragment thereof.

In certain cases of any of formulae 6B to 6D, the sequence fragmentJ¹⁴X¹⁵J¹⁶X¹⁷J¹⁸X¹⁹J²⁰ is IX¹⁵AX¹⁷LX¹⁹(F)_(P) (SEQ ID NO: 37) wherein X¹⁵and X¹⁹ are each independently selected from O and K, X¹⁷ is selectedfrom R and O and p is 1 or 0. In some cases of any one of formulae 6B to6D, the sequence fragment IX¹⁵AX¹⁷LX¹⁹ (F)_(P) (SEQ ID NO: 37) isselected from IOARLK (SEQ ID NO: 38), IOAOLOF (SEQ ID NO: 39) andIKAOLKF (SEQ ID NO: 40). In some cases of any one of formulae 6B to 6D,the sequence fragment IX¹⁵AX¹⁷LX¹⁹(F)_(P)(SEQ ID NO: 37) is IOARLK (SEQID NO: 38). In some cases of any one of formulae 6B to 6D, the sequencefragment IX¹⁵AX¹⁷LX¹⁹ (F)_(P) (SEQ ID NO: 37) is IOAOLOF (SEQ ID NO:39). In some cases of any one of formulae 6B to 6D, the sequencefragment IX¹⁵AX¹⁷LX¹⁹ (F)_(p) is IKAOLKF (SEQ ID NO: 40).

In some cases of the antimicrobial polypeptide of formulae 6, the regionhaving the sequence C-Z-C or GGC-Z-CGG (SEQ ID NO: 25) is referred to asa bubble region as described herein. In certain cases. Z is of theformula J⁹J¹⁰X¹¹J¹², wherein each J is independently selected fromleucine, glycine and phenylalanine and X is selected from ornithine andlysine. In some embodiments, the bubble region in any of formulae 6 to6D has a sequence selected from, CLGOFC (SEQ ID NO: 41), CLGKFC (SEQ IDNO: 42), GGCLGOFC (SEQ ID NO: 43) and GGCLGKFCGG (SEQ ID NO: 44).

Formula 7 Peptides

In some embodiments, the antimicrobial polypeptide comprises a sequencedefined by the formula 7, comprising four cysteine residues:

(SEQ ID NO: 45) Formula 7: Z¹-C¹-Z²-C²-Z³-C³-Z⁴-C⁴-Z⁵wherein, C¹ to C⁴ are each cysteine residues; andZ¹ to Z⁵ are each independently 1-5 amino acid residues. In someembodiments, each of Z¹, Z², Z⁴ and Z⁵ are each striapathic regionsconsisting of a mixture of hydrophobic amino acid residues (J) andhydrophilic residues (X) (e.g., as described herein). Z³ can be anyconvenient linking sequence of residues.

In some embodiments, each of Z¹ to Z⁵ consists of a mixture ofhydrophobic amino acid residues and hydrophilic residues. In someembodiments, each of Z¹ to Z⁴ consists of a mixture of hydrophobicresidues and hydrophilic residues; and Z⁵ is a single hydrophilicresidue. In some embodiments, each of Z² to Z⁵ consists of a mixture ofhydrophobic residues and hydrophilic residues; and Z¹ is a singlehydrophilic residue. In some embodiments, Z¹, Z³ and Z⁵ each consists ofa mixture of hydrophobic residues and hydrophilic residues; and Z² andZ⁴ are each a single hydrophilic residue. In some embodiments Z³consists of a mixture of four amino acid residues selected fromhydrophobic residues and hydrophilic residues. In some embodiments Z²and Z⁴ consist of an equivalent number of amino acid residues.

In some cases, the antimicrobial polypeptide of formula 7 includes oneor more additional hydrophilic or hydrophobic residues at the C terminalor the N terminal.

In some embodiments, the antimicrobial polypeptide comprises a sequencedefined by the formula 7A, based on the formula 7:

Formula 7A: (SEQ ID NO: 46)X¹J²-C¹-J³X⁴J⁵-C²-J⁶X⁷J⁸J⁹-C³-J¹⁰X¹¹X¹²-C⁴⁻X¹³wherein C¹ to C⁴ are each a cysteine residue, each X is independentlyselected from a hydrophilic amino acid residue (e.g. lysine, arginine,histidine, aspartic acid, glutamic acid, asparagine, glutamine andornithine); and each J is independently selected from a hydrophobicresidue (e.g. phenylalanine, tryptophan, alanine, valine, glycine,isoleucine, leucine, and tyrosine). In some cases, each X isindependently selected from lysine, ornithine and arginine. In somecases, each J is independently selected from phenylalanine, isoleucine,glycine, tryptophan, valine and tyrosine.

In some embodiments, the antimicrobial polypeptide of the formula 7A,has the formula 7A¹:

Formula 7A¹: (SEQ ID NO: 47) X¹J²-C¹-J³X⁴V-C²-YX⁷GI-C³-J¹⁰X¹¹X¹²-C⁴-X¹³wherein X¹ is selected from O and R;J² and J³ are each independently selected from F and W;X⁴, X⁷, X¹¹, X¹² and X¹³ are each independently selected from O and K;andJ¹⁰ is selected from Y and F.

In some instances of formula 7A¹, the antimicrobial peptide includes apeptide sequence RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8), or a variantor fragment thereof.

In some instances of formula 7A¹, the antimicrobial peptide includes apeptide sequence RWCFKVCYKGICYKKCK (RP560) (SEQ ID NO: 11), or a variantor fragment thereof.

In some instances of formula 7A′, the antimicrobial peptide includes apeptide sequence OWCFOVCYOGICYOOCO (RP559) (SEQ ID NO: 10), or a variantor fragment thereof.

In some instances of formula 7A¹, the antimicrobial peptide includes apeptide sequence OFCWOVCYOGICFOOCO (RP661) (SEQ ID NO: 12), or a variantor fragment thereof.

In some embodiments, the antimicrobial polypeptide comprises a sequencedefined by the formula 7B, based on the formula 7:

Formula 7B: (SEQ ID NO: 48)X¹-C¹-X²X³J⁴-C²-J⁵J⁶X⁷J⁸-C³-J⁹X¹⁰J¹¹-C⁴-J¹²X¹³wherein C¹ to C⁴ are each a cysteine residue, each X is independentlyselected from a hydrophilic amino acid residue (e.g. lysine, arginine,histidine, aspartic acid, glutamic acid, asparagine, glutamine andornithine); and each J is independently selected from a hydrophobicresidue (e.g. phenylalanine, tryptophan, alanine, valine, glycine,isoleucine, leucine, and tyrosine). In some cases, each X isindependently selected from lysine, ornithine and arginine. In somecases, each J is independently selected from phenylalanine, isoleucine,glycine, tryptophan, valine and tyrosine.

In some embodiments, the antimicrobial polypeptide of the formula 7B,has the formula 7B¹:

Formula 7B¹: (SEQ ID NO: 49) X¹-C¹-X²X³J⁴-C²-IGX⁷Y-C³-VX¹⁰J¹¹-C⁴-J¹²X¹³

wherein X¹, X², X³, X⁷ and X¹⁰ are each independently selected from Oand R;

J⁴ is selected from Y and F;

J¹¹ and J¹² are each independently selected from F and W; and

X¹³ is selected from K and O.

In some instances of formula 7B¹, the antimicrobial peptide includes apeptide sequence RCRRYCIGRYCVRFCWK (RP558) (SEQ ID NO: 9), or a variantor fragment thereof.

In some instances of formula 7B¹, the antimicrobial peptide includes apeptide sequence OCOOFCIGOYCVOWCFO (RP562) (SEQ ID NO: 13), or a variantor fragment thereof.

In some embodiments, the antimicrobial polypeptide comprises a sequencedefined by the formula 7C, based on the formula 7:

Formula 7C: (SEQ ID NO: 50)X¹J²J³-C¹-J⁴-C²-J⁵X⁶X⁷X⁸-C³-J⁹-C⁴-J¹⁰X¹¹J¹²J¹³X¹⁴wherein C¹ to C⁴ are each a cysteine residue, each X is independentlyselected from a hydrophilic amino acid residue (e.g. lysine, arginine,histidine, aspartic acid, glutamic acid, asparagine, glutamine andornithine); and each J is independently selected from a hydrophobicresidue (e.g. phenylalanine, tryptophan, alanine, valine, glycine,isoleucine, leucine, and tyrosine). In some cases, each X is arginine.In some cases, each J is independently selected from phenylalanine,glycine, leucine, valine and tyrosine.

In some embodiments of the antimicrobial polypeptide of formula 7, theregion having the sequence C²-Z³-C³ is a bubble region as describedherein. In certain cases, Z³ consists of a mixture of 3 to 5 hydrophobicand hydrophilic amino acid residues (e.g. J and X as described herein).In certain cases, Z³ consists of a mixture of four amino acid residuesselected from tyrosine, glycine, phenylalanine, isoleucine, lysine,arginine and ornithine. In some cases, Z³ consists of three hydrophobicamino acid residues (J) and one hydrophilic amino acid residue (X). Incertain cases, Z³ consists of a tyrosine, a glycine, an isoleucine and alysine residue. In certain cases, Z³ consists of a tyrosine, a glycine,an isoleucine and an ornithine residue. In certain cases. Z³ consists ofa tyrosine, a glycine, an isoleucine and a lysine residue. In someembodiments, the bubble region in formula 7 has the formula CYKGIC (SEQID NO: 22). In some embodiments, the bubble region in formula 7 has theformula CYOGIC (SEQ ID NO: 51). In some embodiments, the bubble regionin formula 7 has the formula CIGRYC (SEQ ID NO: 52). In someembodiments, the bubble region in formula 7 has the formula CIGOYC (SEQID NO: 53). In some cases, Z³ consists of one hydrophobic amino acidresidue (J) and three hydrophilic amino acid residues (X). In certaincases Z³ consists of one phenylalanine residue and three arginineresidues. In some embodiments, the bubble region in formula 7 has theformula CFRRRC (SEQ ID NO: 54).

In some embodiments of the antimicrobial polypeptide of formula 7, thebubble region having the sequence C²-Z³-C³ forms one linked region, andthe cysteines C¹ and C⁴ link to form an additional linked region (e.g.,see FIG. 1B). In certain embodiments, antimicrobial peptides of formula7 comprises a formula selected from 7A to 7C, each of which form ahairpin secondary structure containing two linked regions. A specificexample of an antimicrobial peptide of the invention according to 7Aincluding a bubble region and an additional linked region includes:

(RP557, SEQ ID NO: 8) RFCWKVCYKGICFKKCK

Examples of antimicrobial peptides according to aspects of the inventionare provided below in Table 2. These examples are representative, andnot meant to be limiting to the scope of the invention. For example, afragment (e.g., as described herein), or a variant (e.g., as describedherein) are also of interest. The “O” residues in the sequences listedbelow represent the amino acid ornithine.

TABLE 2 Examples of antimicrobial peptides SEQ ID RP # NO:Amino Acid Sequence 550  1 FKIOARLCLGOFCIOARLK 551  2FIOKFAKOFKOFIOKFAKFAFAF 552  3 FAFAFKAFKKAFKOFOOAFOOAF 553  4FOIKARFOVRARLOLKI 554  5 FRLKIKARLKVKIRFKL 555  6 FOLOAOIOVOLOAOIOL 556 7 FOLOAOIKVKLOAOIOL 557  8 RFCWKVCYKGICFKKCK 558  9 RCRRYCIGRYCVRFCWK559 10 OWCFOVCYOGICYOOCO 560 11 RWCFKVCYKGICYKKCK 561 12OFCWOVCYOGICFOOCO 562 13 OCOOFCIGOYCVOWCFO 563 14 RGVCVCFRRRCYCLRGGR 56415 FOIOAOLGGCLGOFCGGIOAOLOF 565 16 FOIOAOLOGGCLGOFCGGOIOAOLOF 566 17FOIKAOLGGCLGKFCGGIKAOLKF 567 18 FOIKAOLKGGCLGKFCGGKIKAOLKF

The exemplary antimicrobial polypeptide sequences described herein(e.g., Table 2) are merely examples and are not the only antimicrobialpolypeptides provided herein. Indeed, fragments and variants of thesequences of the disclosed peptides are within the scope of the presentdisclosure.

A “fragment” of the invention includes at least 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous amino acidresidues of a polypeptide disclosed herein (or up to one less than thenumber of amino acid residues in the subject polypeptide) and retains atleast one antimicrobial property of the subject polypeptide. Thus,fragments of the invention include polypeptides that are missing one,two, three, four, or more amino acids from the N-terminus and/or theC-terminus relative to a polypeptide disclosed herein.

A “variant” of the invention is a polypeptide that is substantiallysimilar to a polypeptide disclosed herein and retains at least oneantimicrobial property of the subject polypeptide. Variants can includedeletions (i.e., truncations) of one or more amino acid residues at theN-terminus or the C-terminus of a subject polypeptide disclosed herein;deletion and/or addition of one or more amino acid residues at one ormore internal sites in the subject polypeptide disclosed herein; and/orsubstitution of one or more amino acid residues at one or more positionsin the subject polypeptide disclosed herein. For subject polypeptidesthat are 17 amino acid residues in length or shorter, variantpolypeptides can include three or fewer (e.g., two, one, or none)deleted amino acid residues, whether located internally, at theN-terminal end, and/or at the C-terminal end.

As such, in certain embodiments, the invention provides polypeptidesthat include an amino acid sequence having from 1 to 10 amino aciddifferences (e.g., 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 orfewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino aciddifference) to any one of the antimicrobial polypeptides disclosedherein (e.g., as shown in Table 2) and still retain at least oneantimicrobial property. An “amino acid difference” as used hereinincludes: an amino acid substitution, an amino acid insertion, aterminal amino acid addition, an amino acid deletion, a terminal aminoacid truncation, or any combination thereof. A substituted amino acidresidue (or residues) can be unrelated to the amino acid residue beingreplaced (e.g., unrelated in terms or hydrophobicity/hydrophilicity,size, charge, polarity, etc.), or the substituted amino acid residue canconstitute similar, conservative, or highly conservative amino acidsubstitution. As used herein, “similar,” “conservative,” and “highlyconservative” amino acid substitutions are defined as shown in Table 3,below. The determination of whether an amino acid residue substitutionis similar, conservative, or highly conservative is based exclusively onthe side chain of the amino acid residue and not the peptide backbone,which may be modified to increase peptide stability, as discussed below.

TABLE 3 Classification of Amino Acid Substitutions Highly Amino AcidSimilar Conservative Conservative in Subject Amino Acid Amino Acid AminoAcid Polypeptide Substitutions Substitutions Substitutions Glycine (G)A, S, N A n/a Alanine (A) S, G, T, V, C, P, Q S, G, T S Serine (S) T, A,N, G, Q T, A, N T, A Threonine (T) S, A, V, N, M S, A, V, N S Cysteine(C) A, S, T, V, I A n/a Proline (P) A, S, T, K A n/a Methionine (M) L,I, V, F L, I, V L, I Valine (V) I, L, M, TA I, L, M I Leucine (L) M, I,V, F, T, A M, I, V, F M, I Isoleucine (I) V, L, M, F, T, C V, L, M, F V,L, M Phenylalanine (F) W, L, M, I, V W, L, n/a Tyrosine (Y) F, W, H, L,I F, W F Tryptophan (W) F, L, V F n/a Asparagine (N) Q Q Q Glutamine (Q)N N N Aspartic Acid (D) E E E Glutamic Acid (E) D D D Histidine (H) R,K, O R, K, O R, K, O Lysine (K) R, H, O R, H, O R, O Arginine (R) K, H,O K, H, O K, O Ornithine (O) R, H, K R, H, K R, K

Accordingly, the invention further provides polypeptides that include anamino acid sequence that is at least 50% identical (e.g., at least 60%,70%, 80%, 90%, 95%, 98%, 99% or more identical) to any one of theantimicrobial polypeptides disclosed herein (e.g., as shown in Table 2)and still retain at least one antimicrobial property, in certainembodiment, such polypeptide sequences include an amphipathic orstriapathic region having a cationic charge as described in detailabove. Moreover, such polypeptides may include additional structuralfeatures as described herein, including: a bubble region, a beta-turnregion, a polyproline helix structure, a tail, amphipathic region dimer,etc.

In certain embodiments, the subject antimicrobial polypeptide includes asequence comprising:

-   -   a) a peptide sequence selected from RP550-567 (SEQ ID NO: 1 to        SEQ ID NO 18);    -   b) a sequence having at least 80% sequence identity (e.g., at        least 85%, at least 90%, or at least 95% sequence identity) with        the sequence defined in a); or    -   c) a sequence having five or less (e.g., four or less, three or        less, two or less such as one or two) amino acid substitutions        relative to the sequence defined in a), wherein the one or two        amino acid substitutions are substitutions for amino acids        according to Table 3 (e.g., a similar amino acid substitution, a        conservative amino acid substitution or a highly conservative        amino acid substitution).

In certain embodiments, the subject antimicrobial polypeptide includes asequence comprising:

-   -   a) a peptide sequence selected from RP550-567 (SEQ ID NO: 1 to        SEQ ID NO 18); or    -   b) a sequence having five or less (e.g., four or less, three or        less, two or less such as one or two) amino acid substitutions        relative to the sequence defined in a), wherein the five or less        amino acid substitutions consist of substitution of a cationic        amino acid of the sequence with an alternative cationic amino        acid residue (e.g., K for O, O for K, K for R, etc.).

In certain cases, the sequence defined in a) comprises at least onelysine residue which is substituted for an ornithine, histidine or anarginine residue. In some cases, the sequence defined in a) comprises atleast one lysine residue which is substituted for an ornithine or anarginine residue. In certain other cases, the sequence defined in a)comprises at least one ornithine residue which is substituted for alysine, histidine or an arginine residue. In some cases, the sequencedefined in a) comprises at least one ornithine residue which issubstituted for a lysine or an arginine residue. In other cases, thesequence defined in a) comprises at least one arginine residue which issubstituted for an ornithine, histidine or a lysine residue. In somecases, the sequence defined in a) comprises at least one arginineresidue which is substituted for an ornithine or a lysine residue.

In certain cases, the subject sequence comprises RP 550 (SEQ ID NO: 1).In certain cases, the subject sequence comprises RP 551 (SEQ ID NO: 2).In certain cases, the subject sequence comprises RP 552 (SEQ ID NO: 3).In certain cases, the subject sequence comprises RP 553 (SEQ ID NO: 4).In certain cases, the subject sequence comprises RP 554 (SEQ ID NO: 5).In certain cases, the subject sequence comprises RP 555 (SEQ ID NO: 6).In certain cases, the subject sequence comprises RP 556 (SEQ ID NO: 7).In certain cases, the subject sequence comprises RP 557 (SEQ ID NO: 8).In certain cases, the subject sequence comprises RP 558 (SEQ ID NO: 9).In certain cases, the subject sequence comprises RP 559 (SEQ ID NO: 10).In certain cases, the subject sequence comprises RP 560 (SEQ ID NO: 11).In certain cases, the subject sequence comprises RP 561 (SEQ ID NO: 12).In certain cases, the subject sequence comprises RP 562 (SEQ ID NO: 13).In certain cases, the subject sequence comprises RP 563 (SEQ ID NO: 14).In certain cases, the subject sequence comprises RP 564 (SEQ ID NO: 15).In certain cases, the subject sequence comprises RP 565 (SEQ ID NO: 16).In certain cases, the subject sequence comprises RP 566 (SEQ ID NO: 17).In certain cases, the subject sequence comprises RP 567 (SEQ ID NO: 18)

In certain cases, the sequence set forth in a) is RP 550 (SEQ ID NO: 1).In certain cases, the sequence set forth in a) is RP 551 (SEQ ID NO: 2).In certain cases, the sequence set forth in a) is RP 552 (SEQ ID NO: 3).In certain cases, the sequence set forth in a) is RP 553 (SEQ ID NO: 4).In certain cases, the sequence set forth in a) is RP 554 (SEQ ID NO: 5).In certain cases, the sequence set forth in a) is RP 555 (SEQ ID NO: 6).In certain cases, the sequence set forth in a) is RP 556 (SEQ ID NO: 7).In certain cases, the sequence set forth in a) is RP 557 (SEQ ID NO: 8).In certain cases, the sequence set forth in a) is RP 558 (SEQ ID NO: 9).In certain cases, the sequence set forth in a) is RP 559 (SEQ ID NO:10). In certain cases, the sequence set forth in a) is RP 560 (SEQ IDNO: 11). In certain cases, the sequence set forth in a) is RP 561 (SEQID NO: 12). In certain cases, the sequence set forth in a) is RP 562(SEQ ID NO: 13). In certain cases, the sequence set forth in a) is RP563 (SEQ ID NO: 14). In certain cases, the sequence set forth in a) isRP 564 (SEQ ID NO: 15). In certain cases, the sequence set forth in a)is RP 565 (SEQ ID NO: 16). In certain cases, the sequence set forth ina) is RP 566 (SEQ ID NO: 17). In certain cases, the sequence set forthin a) is RP 567 (SEQ ID NO: 18).

In certain instances of the subject peptides (e.g., described herein)and compositions, one or more of the following peptide sequences ofTable 4 are excluded:

TABLE 4 SEQ ID NO: Amino Acid Sequence 56 RVFKKAFRKFKKLFKRAF 57FARKFLKKFKRFAKKFVR 58 FKRKIKAKLRFKAKVRLK 59 FAFAFRVFKKAFRKFKKLFKRAF 60FARKFLKKFKRFAKKFVRFAFAF 61 KIRAKLCLGRFCIRAKLR 62 KIKARLCLGKFCIKARLK 63FAFAFKAFKKAFKKFKKAFKKAFGPGRFAKKFAKKFKKFAKKFAK FAFAF 64FAKKFAKKFKKFAKKFAKFAFAFGPGRFAFAFKAFKKAFKKFKKA FKKAF 65MGFKLRAKIKVRLRAKIKL 66 CVOLFPVOLFPC 67 CKLRFRGPGRIKVRLC 68CPGFAKKFAKKFKKFAKKFAKFAFAF 69 KIRAKLCLGRFCIRAKLR 70KKKPKPPYLPKPKPPPFFPPKLPPKI 71 FAFAFKAFKKAFKKFKKAFKKAFGPC 72FAFAFAFKKAFKKFKKAFKKAF 73 FAFAFOAFOOAFOOFOOAFOOAF 74FAOOFAOOFOOFAOOFAOFAFAF 75 FAKKFAKKFKKFAKKFAFAFAF 76RLARIVGGFAOOFAOOFOOFAOOFAOFAFAF 77 CRLARIVCGGFAOOFAOOFOOFAOOFAOFAFAF 78FOIOAOLGGCLGOFCGGIOAOLOF 79 OLOSLLKTLSOAOOOOLOTOOOAISO 80ALWMTLOOOVLOAOAOALNAVLVGANA 81 AFAFTAOOOFAOFOAOFANFAFAGFNACompositions

The present disclosure provides compositions that include anantimicrobial polypeptide as described herein. For example, theantimicrobial polypeptide can be any of the polypeptides listed in Table2 or a fragment or variant thereof that retains antimicrobial activity.In certain embodiments, the antimicrobial polypeptide included in thecompositions of this disclosure will be a synthetic polypeptide (e.g.,made by chemical synthesis and/or produced recombinantly).

The compositions of the invention can include a single antimicrobialpolypeptide, or combinations of different antimicrobial polypeptides.The compositions can be substantially free of proteins and otherpolypeptides. As used herein, the term “substantially free of proteinsand other polypeptides” means that less than 5% of the protein contentof the composition is made up of proteins and other polypeptides thatare not an antimicrobial polypeptide of the invention. A compositionthat is substantially free of non-antimicrobial polypeptides of theinvention can have less than 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%,or less of other non-antimicrobial polypeptides.

The compositions of the invention in certain embodiments contain anantimicrobial polypeptide that is not naturally found in a human orother mammal or animal.

The subject compositions can be provided in any convenient form. In somecases, the composition is a solid composition. In certain cases, thecomposition is a liquid, e.g., aqueous composition. In certaininstances, the solid composition is a lyophilized composition that canbe provided to an end use in a solid form suitable for reconstitutionwith a liquid, e.g., an aqueous solution.

The compositions of the invention can include at least 0.1 mg (e.g., atleast 0.2, at least 0.3, at least 0.4, at least 0.5, at least 1, atleast 5, at least 10, at least 20, at least 30, at least 40, at least50, at least 75, at least 100, at least 150, at least 200, at least 250,at least 300, at least 400, at least 500, at least 600, at least 700, atleast 800, at least 900, at least 1000 mg, or more) of antimicrobialpolypeptide. Thus, for example, the compositions can include an amountof antimicrobial polypeptide equal to about 0.1 mg to about 1 mg, orabout 1 mg to about 1000 mg (e.g., about 5 mg to about 900 mg, about 5mg to about 800 mg, about 5 mg to about 700 mg, about 5 mg to about 600mg, about 10 mg to about 500 mg, about 10 mg to about 400 mg, about 10mg to about 300 mg, about 10 mg to about 250 mg, about 10 mg to about200 mg, about 10 mg to about 150 mg, about 10 mg to about 100 mg, about50 mg to about 500 mg, about 50 mg to about 400 mg, about 50 mg to about300 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about50 mg to about 150 mg, about 50 mg to about 100 mg, about 75 mg to about500 mg, about 75 mg to about 400 mg, about 75 mg to about 300 mg, about75 mg to about 250 mg, about 75 mg to about 200 mg, about 75 mg to about150 mg, about 75 mg to about 100 mg, about 100 mg to about 500 mg, about100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg toabout 250 mg, about 100 mg to about 200 mg, or any other rangecontaining two of the foregoing endpoints).

The compositions of the invention can include a solution that containsat least 0.1 mg/ml (e.g., at least 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/ml ormore) of an antimicrobial polypeptide. Thus, for example, thecompositions can include a solution having an antimicrobial polypeptideconcentration of about 0.1 mg/ml to about 100 mg/ml (e.g., about 5 mg/mlto about 90 mg/ml, about 5 mg/ml to about 80 mg/ml, about 5 mg/ml toabout 70 mg/ml, about 5 mg/ml to about 60 mg/ml, about 5 mg/ml to about50 mg/ml, about 10 mg/ml to about 50 mg/ml, about 10 mg/ml to about 40mg/ml, about 10 mg/ml to about 30 mg/ml, about 10 mg/ml to about 25mg/ml, about 10 mg/ml to about 20 mg/ml, about 10 mg/ml to about 15mg/ml, or any other range containing two of the foregoing endpoints).

The compositions of the invention include pharmaceutical compositions.Such pharmaceutical compositions can comprise one or more antimicrobialpolypeptides and a pharmaceutically acceptable carrier. Pharmaceuticalcompositions can further include an additional bioactive agent otherthan an antimicrobial polypeptide of the invention. The additionalbioactive agent can be a therapeutic/antimicrobial agent, such as aconventional antibiotic. The conventional antibiotic can haveantimicrobial properties or other properties that the antimicrobialpolypeptides of the invention augment or are augmented by. In someembodiments, the additional bioactive agent is selected from anantimicrobial agent, an anti-inflammatory drug, an anti-nausea drug, ananti-pain medication and combinations thereof. In certain embodimentsthe pharmaceutical composition includes a carrier, e.g., a carrierprotein such as serum albumin (e.g., HAS, BSA, etc.), which can bepurified or recombinantly produced. By mixing the antimicrobialpolypeptide(s) in the pharmaceutical composition with serum album, theantimicrobial polypeptides can be effectively “loaded” onto the serumalbumin, allowing a greater amount of antimicrobial polypeptide to besuccessfully delivered to a site of infection.

The pharmaceutical compositions of the present invention can beformulated for oral administration, parenteral administration,inhalation administration, topical administration, or the like.Compositions formulated for oral delivery can, for example, include anenteric coat, to ensure that antimicrobial peptides contained thereinreach the intestine and beyond. Compositions formulated for topicaldelivery can be, for example, suspended in a gel or cream, coated on amicroneedle, or infused into a bandage or topical patch, to extend theduration of action of the antimicrobial peptides contained therein. Anyinhalable formulation which can provide for an aerosolized formincluding a subject peptide for delivery to a patient via theintrapulmonary route may be used in conjunction with the presentdisclosure. In some cases, an inhalable composition includes liposomeswhich are delivered via an aerosol to the lungs of a human patient, theliposomes comprising free and encapsulated peptide. The liposomes may beunilamellar or multilamellar, and may be bioadhesive, containing amolecule such as hyaluronic acid. At least one therapeutic agent inaddition to the free and liposome-encapsulated anti-infective may alsobe included in the composition. The therapeutic agent may be freepeptide or encapsulated peptide present with a pharmaceuticallyacceptable carrier useful for direct inhalation into human lungs.

In certain embodiments, it may be desirable to administer one or morecompounds of the invention locally to the area in need of treatment.This may be achieved, for example, by local infusion during surgery,topical application, e.g., in conjunction with a wound dressing aftersurgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers.

In certain instances, the pharmaceutical composition is an ophthalmiccomposition formulated for the treatment of an eye disease or condition.The ophthalmic compositions described herein may be formulated in anyapplicable dosage form. Exemplary dosage forms include, but are notlimited to, eye drops (liquids), ointments, oils, multi-phase systems(such as, liposome, micellular, homogenates or suspensions of liquids orsemi-solid or solid particles), gels, creams, pads or ships. In oneembodiment, the active ingredient (peptide) is in a water-based(aqueous) drug product. In another embodiment, the active ingredient isin a petrolatum-based drug product. One embodiment of the presentinvention is the use of topical formulations of peptides describedherein to treat ocular infections caused by, without limitation,bacteria, herpes simplex virus, cytomegalovirus, varicella zoster virus,adenovirus and/or a combination thereof. One embodiment of the presentinvention is the use of topical formulations of peptides describedherein to treat bacterial keratitis. In some cases, the bacterialkeratitis is caused by infection with Staphylococcus aureus orPseudomonas aeruginosa.

The composition can be topically applied to a patient in need of thetreatment. In some cases, the composition is formulated as eye drops. Incertain cases, the composition is formulated for subconjunctivaladministration. In some cases, the ophthalmic composition is an aqueouscomposition which includes the subject peptide or a pharmacologicallyacceptable salt, optionally in combination with an amount of anyconvenient additional excipients, e.g., a cellulose compound or apharmacologically acceptable salt which is effective to increaseintraocular absorption in the aqueous humor. The ophthalmic compositionmay be applied discretely one to four times per day as eye drops.Alternatively, the antimicrobial peptide may be continuously appliedover time by any topical means. A hydrophilic hydrogel contact lens andan ocular polymer insert for topical application of an ophthalmiccomposition to the eyes of a patient is also disclosed. Any convenientmethods and compositions can be adapted for use in conjunction with thesubject ophthalmic compositions. See e.g., U.S. Pat. No. 9,044,425. Thecomposition can include any carrier or excipients that known to oneskilled in the art that are suitable for intraocular administration.Exemplary excipients include those discussed in U.S. Patent ApplicationPublication No. 2008/0241252 to Lyons et al. and PCT ApplicationPublication No. WO 2004/043480, which are incorporated by references intheir entireties.

Although liposomes have been described as a vehicle to provideencapsulation of the therapeutic peptide, there is no intention to limitthe formulation to liposomal formulations. A combination of immediateand sustained release formulations or carriers of an anti-infectivepeptide in the lung may be achieved via a multitude of ways includingmicrospheres, polymers, gels, emulsions, particulates or suspensions,either singly or in combination. Some formulations or carriers may haveproperties that result in closer association with the biofilm matrix andthese may prove more advantageous with respect to increasing thetherapeutic levels of the anti-infective peptide proximal to the biofilmbacteria.

Aspects of the disclosure include antimicrobial compositions that finduse in a variety of applications. In some instances, the subjectcomposition finds use as an antimicrobial preservative, disinfectant orsterile storage medium. Applications of interest include use as adisinfectant or storage medium for an ocular device, such as a contactlens, an intraocular lens implant or a drug-eluting ocular device, incertain instances, the subject composition can be provided to a subject,as a solid or liquid composition, for use in a solution for storing,soaking and/or rinsing a contact lens. The solutions are well suited forophthalmic use and are effective in cleaning, disinfecting, andsterilizing the contact lens upon exposure to the composition withoutthe need for physical or thermal treatment of the lens. Any convenientcarriers, excipients and/or diluents that find use in sterile ordisinfecting solutions can be adapted for inclusion in the subjectantimicrobial compositions. See e.g., U.S. Pat. No. 6,482,799.

Alternatively, the antimicrobial peptides of the present disclosure canbe coated on the surface of medical devices, such as implantable medicaldevices, surgical instruments and indwelling medical devices (e.g.,pacemakers, catheters, artificial joints, and the like), as a means ofpreventing infection.

Methods

The polypeptides of this disclosure provide powerful tools for treatingor preventing a variety of conditions and diseases of interest. Aspectsof the method include administering to a subject in need thereof atherapeutically effective amount of a subject peptide to treat thesubject. By “a therapeutically effective amount” is meant theconcentration of a peptide that is sufficient to elicit the desiredbiological effect (e.g., treatment of the condition or disease). Theterms “treat,” “treating,” and similar words shall mean stabilizing,reducing the symptoms of, preventing the occurrence of, or curing amedical condition. As such, treatment also includes situations where thepathological condition, or at least symptoms associated therewith, arecompletely inhibited, e.g., prevented from happening, or stopped, e.g.terminated, such that the host no longer suffers from the condition, orat least the symptoms that characterize the condition. Thus treatmentincludes: (i) prevention, that is, reducing the risk of development ofclinical symptoms, including causing the clinical symptoms not todevelop, e.g., preventing disease progression to a harmful state; (ii)inhibition, that is, arresting the development or further development ofclinical symptoms, e.g., mitigating or completely inhibiting an activedisease; and/or (iii) relief, that is, causing the regression ofclinical symptoms.

The antimicrobial polypeptides of the invention provide powerful toolsfor treating or preventing a microbial infection in a subject, caused bya variety of microorganisms (e.g., bacteria, viruses, fungi, andparasites). Accordingly, the invention provides methods of eliminating,reducing the number of, or significantly reducing the replication of atleast one microbial organism in a subject. The subject peptides can havebroad-spectrum antifungal and antibacterial activity. In certain cases,the subject peptides and methods provide a reduced risk of developmentof pathogen resistance.

The subject can be any animal, such as a domesticated animal (e.g., ahorse, cow, pig, goat, sheep, rabbit, chicken, turkey, duck, etc.), apet (e.g., a dog, cat, rabbit, hamster, gerbil, bird, fish, etc.), a labanimal (e.g., a mouse, rat, monkey, chimpanzee, owl, fish, etc.), a zooanimal (e.g., a gorilla, orangutan, chimpanzee, monkey, elephant, camel,zebra, boar, lion, tiger, giraffe, bear, bird, etc.), a wild animal(e.g., a deer, wolf, mountain lion, bird, etc.), or a human subject(e.g., a patient). In some cases, the subject is human.

A subject suitable for treatment with a peptide composition may beidentified by well-established indicators of risk for developing adisease or well-established hallmarks of an existing disease. Forexample, indicators of an infection include fever, pus, microorganismpositive cultures, inflammation, and the like. Infections that may betreated with peptides provided by the present invention include withoutlimitation those caused by or due to microorganisms, whether theinfection is primary, secondary, opportunistic, or the like. Examples ofmicroorganisms include bacteria (e.g., Gram-positive, Gram-negative),fungi, (e.g., yeast and molds), parasites (e.g., protozoans, nematodes,cestodes and trematodes), viruses (e.g., HIV, HSV, VSV), algae, andprions. Specific organisms in these classes are well known (see, forexample, Davis et al., Microbiology, 3 rd edition, Harper & Row, 1980,and Stanier et al., The Microbial World, 5th edition, Prentice Hall,1986). Infections of interest that may be treated or prevented accordingto the subject methods include, hut are not limited to, toxic shocksyndrome, diphtheria, cholera, typhus, meningitis, whooping cough,botulism, tetanus, pyogenic infections, sinusitis, pneumonia,gingivitis, mucitis, folliculitis, cellulitis, acne and acne vulgaris,impetigo, osteomyelitis, endocarditis, ulcers, burns, dysentery, urinarytract infections, gastroenteritis, anthrax, Lyme disease, syphilis,rubella, septicemia, Buruli ulcer, mycetoma, chromoblastomycosis,vaginal candidiasis, tuberculosis, otitis media, eczema (atopicdermatitis), diabetic ulcers, impetigo, toenail fungus, venous ulcers,infected burns, infected wounds, infected ballistic wounds and plague;as well as primary, secondary, and opportunistic infections associatedwith, for example, trauma, surgery, endotracheal intubation,tracheostomy, and cystic fibrosis.

A subject may have other clinical indications that have associatedinfection or inflammation treatable or preventable with the compositionsand methods of the present invention, which include without limitationthose associated with implantable, indwelling, or similar medicaldevices, such as intravascular catheters (e.g., intravenous andintraarterial), right heart flow-directed, catheters, Hickman catheters,arteriovenus fistulae, catheters used in hemodialysis and peritonealdialysis (e.g., silastic, central venous. Tenckhoff, and tefloncatheters), vascular access ports, indwelling urinary catheters, urinarycatheters, silicone catheters, ventricular catheters, synthetic vascularprostheses (e.g., aortofemoral and femoropopliteal), prosthetic heartvalves, prosthetic joints, orthopedic implants, penile implants, shunts(e.g., Scribner, Torkildsen, central nervous system, portasystemic,ventricular, ventriculoperitoneal), intrauterine devices, tampons,contact lenses, dental implants, ureteral stents, pacemakers,implantable defibrillators, tubing, cannulas, probes, blood monitoringdevices, needles, and the like. A “medical device” refers to any devicefor use in a subject, such as an animal or human.

The amount of the subject peptide composition administered can bedetermined using any convenient methods to be an amount sufficient toproduce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for the unitdosage forms of the present disclosure will depend on the particularcompound employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

The antimicrobial polypeptide(s) can be administered at a dose andfrequency that depends on the type of animal, the size of the animal,and the condition being treated. It should also be understood that aspecific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the activity of the specificcompound employed, the mode of administration, the age, body weight,general health, gender, diet, rate of excretion, drug combination, andthe judgment of the treating physician, the condition being treated andthe severity of the condition. Such dosage may be ascertained readily bya person skilled in the art. This dosage regimen may be adjusted toprovide the optimal therapeutic response. In some cases, theantimicrobial polypeptide is administered daily (or every other day, orweekly), in an amount between about 1 mg and about 1000 mg (e.g., about5 mg to about 900 mg, about 5 mg to about 800 mg, about 5 mg to about700 mg, about 5 mg to about 600 mg, about 10 mg to about 500 mg, about10 mg to about 400 mg, about 10 mg to about 300 mg, about 10 mg to about250 mg, about 10 mg to about 200 mg, about 10 mg to about 150 mg, about10 mg to about 100 mg, about 50 mg to about 500 mg, about 50 mg to about400 mg, about 50 mg to about 300 mg, about 50 mg to about 250 mg, about50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about100 mg, about 75 mg to about 500 mg, about 75 mg to about 400 mg, about75 mg to about 300 mg, about 75 mg to about 250 mg, about 75 mg to about200 mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg toabout 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200mg, or any other range containing two of the foregoing endpoints). Thedaily dose can be administered once during the day, or broken up intosmaller doses that are taken at multiple time points during the day. Fora human (and other similarly-sized mammals), a dose of 5 mg/kg everyother day can be administered. The antimicrobial polypeptide can beadministered for a fixed period of time (e.g., for 2-3 weeks), atintervals (e.g., administer polypeptide for 2-3 weeks, wait 2-3 weeks,then repeat the cycle), or until such time as the microbial organism hasbeen eliminated or significantly reduced, the symptoms of the microbialinfection have been ameliorated, or the potential microbial infectionrisk has been reduced or eliminated (e.g., a wound has healed).

For intrapulmonary administration, a patient can receive a dose of about0.01 to 10 mg/kg/day of the peptide ±20% or ±10%. This dose can beadministered by at least one, preferably several “puffs” from an aerosoldevice. The total dose per day is preferably administered at least onceper day, but may be divided into two or more doses per day. Somepatients may benefit from a period of “loading” the patient with thesubject peptide with a higher dose or more frequent administration overa period of days or weeks, followed by a reduced or maintenance dose.

The administration of the antimicrobial polypeptides (or pharmaceuticalcompositions comprising such polypeptides) in conjunction with any ofthe foregoing methods can be performed intravenously, intraperitoneally,parenterally, orthotopically, subcutaneously, topically, via inhalation,nasally, orally, sublingually, intraocularly, by means of an implantabledepot, using nanoparticle-based delivery systems, microneedle patch,microspheres, beads, osmotic or mechanical pumps, and/or othermechanical means. In some embodiments, the pharmaceutical compositionsof the invention can be administered to a subject by applying thecomposition to a surface of a medical device prior to inserting themedical device into the subject. Systemic administration can be achievedvia intravenous, intramuscular or subcutaneous injections or infusions.In certain cases, the subject peptide compositions are administeredorally. In certain instances, the subject peptide compositions areadministered via inhalation. In some cases, the subject peptidecompositions are administered topically.

One aspect of the present disclosure is to provide methods for treatingor preventing a infection of the eye by any pathogen of interest (e.g.,as described herein), such as a viral or bacterial infection. In someembodiments, the methods comprise administering a pharmaceuticallyacceptable composition to the ocular region of a subject. Any convenientophthalmic delivery route can be utilized in conjunction with thesubject methods and compositions, including but not limited to, topicalinstillation, subconjunctival, subtenon, intravitreal, retrobulbar, andintracameral administration. For example, the composition may betopically applied (e.g. as eye drops, via a hydrophilic hydrogel contactlens, or via an ocular polymer insert for topical application) to theeye. The pharmaceutically acceptable composition comprises apharmaceutically acceptable carrier and at least one peptide asdescribed herein. In another embodiment, the composition may be orallyadministered to the subject. Intraocular administration of a subjectophthalmic composition can be achieved via subconjunctival (into thesubconjunctival), intravitreal (into the vitreous), subretinal (underthe retina), or retrobulbar (behind the eyeball) injection.

The eye infections described herein may be any applicable pathogen thatcan infect the eye, such as bacteria, fungi and viruses. Exemplaryinfections include, but are not limited to, bacterial conjunctivitis(e.g., caused by Haemophilus influenza. Streptococcus pneumoniae orStaphylococcus aureus), Gonococcal conjunctivitis, Chlamydialconjunctivitis, fungal keratitis, trachoma, bacterial endophthalmitis,bacterial keratitis, and the like.

The viral infection described herein may be any applicable virus thatcan infect the eye, such as viral keratitis. Exemplary viral infectionsinclude, but are not limited to, viral conjunctivitis, influenza, herpessimplex virus (HSV), human herpes virus, cytomegalovirus (CMV),Epstein-Barr virus (EBV), varicella zoster virus (VZV), orthopox virus,variola major and minor, vaccinia, cowpox, camelpox, monkeypox,papilloma virus, adenovirus, polyoma virus including JC virus, BK virus,SV40 and a combination thereof. In some embodiments, at least one viralinfection is selected from cytomegalovirus, varicella zoster virus,adenovirus, herpes simplex virus and Epstein-Barr virus. In anotherembodiment, the viral infection is a topical viral infection.

Another aspect of the invention is to provide methods for treatment orpreventing a posterior ocular condition. In some embodiments, posteriorocular condition or degeneration condition of retina or retinal nerve,is selected from macular degeneration, retinopathy, or retinitispigmentosa. Further, in one embodiment, the pharmaceutical compositionfor treating posterior ocular condition is intraocular administered (forexample, intraocular injection including retrobulbar, intravitreal,intraretinal, intracameral or subconjunctival injection).

Infection is the predominant determinant of wound healing, incidence ofcomplications, and outcome of burn patients. The main organismsresponsible are Pseudomonas aeruginosa, S. aureaus, Streptococcuspyogenes, and various gram-negative organisms. Frequent debridements andestablishment of an epidermis or a surrogate, such as a graft or a skinsubstitute, is essential for prevention of infection. In some cases, thesubject peptide composition, alone or in combination with antibioticand/or anti-inflammatory agents, is applied to burn wounds as a gel,ointment or cream, and/or administered systemically. Topical applicationcan prevent systemic infection following superficial colonization oreradicate a superficial infection. In some cases, the subjectcomposition can be administered as a 0.5% to 5% gel, cream, or ointment,such as 0.5 to 2%, as described herein. Application to the skin could beperformed once a day or as often as dressings are changed. Systemicadministration can be achieved via intravenous, intramuscular orsubcutaneous injections or infusions. Other routes of administration canalso be used.

The compositions and methods of the present disclosure also find use inthe treatment of nosocomial infections. For example, infection by S.aureus may result in impetigenous lesions or infected wounds and isassociated with increased infection rates following cardiac surgery,hemodialysis, orthopedic surgery and neutropenia, both disease-inducedand iatrogenic. Nasal and extra-nasal carriage of Staphylococci spp. canresult in hospital outbreaks of the same Staphylococci strain that iscolonizing a patient's or a hospital worker's nasal passage orextra-nasal site.

The compositions and methods of the present disclosure also find use inthe treatment of acute bacterial skin and skin structure infections(ABSSSI). Also of interest are methods of treating gram-negativepathogens and multidrug-resistant gram-positive bacteria, such ascommunity-acquired methicillin resistant Staphylococcus aureus (MRSA).Gram-negative bacteria of interest that may be targeted according to thesubject methods include, but are not limited to, A. baumannii and P.aeruginosa; gram-positive bacteria, S. aureus and MRSA and fungalstrains; C. albicans, C. parapsilosis, C. krusei, Aspergillus fumigatus,Aspergillus flavus, Absidia corymbifera, Fusarium solani and Mucor.

In some case, the subject being treating according to the subjectmethods is infected with an antibiotic-resistant microorganism selectedfrom a gram positive bacterium, a gram negative bacterium, abiofilm-forming bacterium, Streptococcus pneumoniae, Campylobacter,Neisseria gonorrhoeae, Salmonella, Methicillin-resistant Staphylococcusaureus (MRSA), Shigella. Vancomycin-resistant Enterococcus (VRE),Vancomycin-resistant Staphylococcus aureus (VRSA).Erythromycin-resistant Group A Streptococcus, Clindamycin-resistantGroup B Streptococcus, Carbapenem-resistant Enterobacteriaceae (CRE),drug-resistant tuberculosis, Extended spectrum Enterobacteriaceae(ESBL), multidrug-resistant Acinetobacter (including MRAB), Clostridiumdifficile, Enteropathogenic E. coli (EPEC), Pseudomonas aeruginosa, H.pylori, Streptococcus anginosus and Uropathogenic E. coli (UPEC), S.epidermidis, E. faecalis, E. coli, S. aureus, Campylobacter, andPseudomonas or combinations thereof.

The compositions and methods of the present disclosure also find use inthe treatment or inhibition of a biofilm. “Inhibition” or “inhibiting”of biofilm formation refers to a decrease of biofilm associatedmicroorganism formation and/or growth. The subject compositions areeffective against biofilms produced by a wide range of microbial speciesincluding, but not limited to, S. epidermidis, E. faecalis, E. coli, S.aureus, Campylobacter spp. H. pylori and Pseudomonas, alone, or incombination. The biofilm can be associated with a bacterial infectionselected from impetigo, boils, abscesses, folliculitis, cellulitis,necrotizing fasciitis, pyomyositis, surgical/traumatic wound infection,and infected ulcers and burns, osteomyelitis, device-relatedosteoarticular infections, impetigo, secondarily infected skin lesions,meningitis, brain abscess, subdural empyema, spinal epidural abscess,arterial damage, gastritis, urinary tract infections, biliary tractinfections, pyelonephritis, cystitis, sinus infections, ear infections,otitis media, otitis externa, leprosy, tuberculosis, conjunctivitis,bloodstream infections, benign prostatic hyperplasia, chronicprostatitis, lung infections, osteomyelitis, catheter infections,bloodstream infections, skin infections, acne, rosacea, dental caries,periodontitis, gingivitis, nosocomial infections, arterial damage,endocarditis, periprosthetic joint infections, open or chronic woundinfections, venous stasis ulcers, diabetic ulcers, arterial leg ulcers,pressure ulcers, endocarditis, pneumonia, orthopedic prosthesis andorthopedic implant infections, peritoneal dialysis peritonitis,cirrhosis, and other acute or chronic infection that involves orpossesses a biofilm.

In some embodiments, the subject antimicrobial peptide hasanti-inflammatory activity. The present disclosure also provides amethod of treating a condition associated with cancer. Cancers ofinterest include solid tumor cancers. In such methods, a pharmaceuticalcomposition including a subject peptide (e.g., as described herein) canbe administered locally at a site of interest, e.g., intra-tumorally viainjection, or the like.

In conjunction with any of the foregoing methods, the antimicrobialpolypeptides (or pharmaceutical compositions comprising suchpolypeptides) can be administered in combination with another drug,e.g., an antibiotic, antiviral, antifungal, antiprotozoal, antimalarial,or a drug for treating a non-infectious disease or other condition. Incertain embodiments, the other drug is one that can reduce a symptom ofa disease/microbial infection (e.g., to reduce or prevent a fever, totreat or prevent nausea, etc.). In each case, the antimicrobialpolypeptide can be administered prior to, at the same time as, or afterthe administration of the other drug.

As noted above, the subject antimicrobial peptides may be used in asynergistic combination with an additional antimicrobial agent.Antibacterial agents of interest include, but are not limited to,penicillins, cephalosporins, carbacephems, cephamycins, carbapenems,monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines,macrolides, and fluoroquinolones. Examples of antibiotic agents include,but are not limited to, Penicillin G (CAS Registry No.: 61-33-6);Methicillin (CAS Registry No.: 61-32-5); Nafcillin (CAS Registry No.:147-52-4); Oxacillin (CAS Registry No.: 66-79-5); Cloxacillin (CASRegistry No.: 61-72-3); Dicloxacillin (CAS Registry No.: 3116-76-5);Ampicillin (CAS Registry No.: 69-53-4); Amoxicillin (CAS Registry No.:26787-78-0); Ticarcillin (CAS Registry No.: 34787-01-4); Carbenicillin(CAS Registry No.: 4697-36-3); Mezlocillin (CAS Registry No.:51481-65-3); Azlocillin (CAS Registry No.: 37091-66-0); Piperacillin(CAS Registry No.: 61477-96-1); Imipenem (CAS Registry No.: 74431-23-5);Aztreonam (CAS Registry No.: 78110-38-0); Caphalothin (CAS Registry No.:153-61-7); Cefazolin (CAS Registry No.: 25953-19-9); Cefaclor (CASRegistry No.: 70356-03-5); Cefamandole formate sodium (CAS Registry No.:42540-40-9); Cefoxitin (CAS Registry No.: 35607-66-0); Cefuroxime (CASRegistry No.: 55268-75-2); Cefonicid (CAS Registry No.: 61270-58-4);Cefmetazole (CAS Registry No.: 56796-20-4); Cefotetan (CAS Registry No.:69172-56-7); Cefprozil (CAS Registry No.: 92665-29-7); Loracarbef (CASRegistry No.: 121961-22-6) Cefetamet (CAS Registry No.: 65052-63-3);Cefoperazone (CAS Registry No.: 62893-19-0); Cefotaxime (CAS RegistryNo.: 63527-52-6); Ceftizoxime (CAS Registry No.: 68401-81-0) Ceftriazone(CAS Registry No.: 73384-59-5); Ceftazidime (CAS Registry No.:72558-82-8); Cefepime (CAS Registry No.: 88040-23-7); Cefixime (CASRegistry No.: 79350-37-1); Cefpodoxime (CAS Registry No.: 80210-62-4);Cefsulodin (CAS Registry No.: 62587-73-9); Fleroxacin (CAS Registry No.:79960-72-3); Nalidixic acid (CAS Registry No.: 389-08-2); Norfloxacin(CAS Registry No.: 70458-96-7); Ciprofloxacin (CAS Registry No.:85721-33-1); Ofloxacin (CAS Registry No.: 82419-36-1); Enoxacin (CASRegistry No.: 74011-58-8); Lomefloxacin (CAS Registry No.: 98079-51-7);Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry No.:564-25-0); Minocycline (CAS Registry No.: 10118-90-8); Tetracycline (CASRegistry No.: 60-54-8); Amikacin (CAS Registry No.: 37517-28-5);Gentamicin (CAS Registry No.: 1403-66-3); Kanamycin (CAS Registry No.:8063-07-8); Netilmicin (CAS Registry No.: 56391-56-1); Tobramycin (CASRegistry No.: 32986-56-4); Streptomycin (CAS Registry No.: 57-92-1);Azithromycin (CAS Registry No.: 83905-01-5); Clarithromycin (CASRegistry No.: 81103-11-9); Erythromycin (CAS Registry No.: 114-07-8);Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin ethylsuccinate (CAS Registry No.: 41342-53-4); Erythromycin glucoheptonate(CAS Registry No.: 23067-13-2); Erythromycin lactobinate (CAS RegistryNo.: 3847-29-8); Erythromycin stearate (CAS Registry No.: 643-22-1);Vanomycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.:61036-64-4); Chloramphenicol (CAS Registry No.: 56-75-7); Clindamycin(CAS Registry No.: 18323-44-9); Trimethoprim (CAS Registry No.:738-70-5); Sulfamethoxazole (CAS Registry No.: 723-46-6); Nitrofurantoin(CAS Registry No.: 67-20-9); Rifampin (CAS Registry No.: 13292-46-1);Mupirocin (CAS Registry No.: 12650-69-0); Metronidazole (CAS RegistryNo.: 443-48-1); Cephalexin (CAS Registry No.: 15686-71-2); Roxithromycin(CAS Registry No.: 80214-83-1); Co-amoxiclavunate; combinations ofPiperacillin and Tazobactam; and their various salts, acids, bases, andother derivatives, and combinations thereof.

The subject antimicrobial peptide may also be used in combination withan anti-fungal agents. Exemplary anti-fungal agents of interest include,but are not limited to, terbinafine hydrochloride, nystatin,amphotericin B, griscofulvin, ketoconazole, miconazole nitrate,flucytosine, fluconazole, itraconazole, clotriamzole, benzoic acid,salicylic acid, and selenium sulfide.

The subject antimicrobial peptide may also be used in combination withan anti-viral agent. Exemplary anti-viral agents of interest include,but are not limited to, amantadine hydrochloride, rimantadin, acyclovir,famciclovir, foscamet, ganciclovir sodium, idoxuridine, ribavirin,sorivudine, trifluoridine, valacyclovir, vidarabin, didanosine,stavudine, zalcitabine, zidovudine, interferon alpha, and edoxudine.

The subject antimicrobial peptide may also be used in combination withan anti-parasitic agent. Exemplary anti-parasitic agents of interestinclude, but are not limited to, pirethrins/piperonyl butoxide,permethrin, iodoquinol, metronidazole, diethylcarbamazine citrate,piperazine, pyrantel pamoate, mebendazole, thiabendazole, praziquantel,albendazole, proguanil, quinidine gluconate injection, quinine sulfate,chloroquine phosphate, mefloquine hydrochloride, primaquine phosphate,atovaquone, co-trimoxazole (sulfamethoxazole/trimethoprim) andpentamidine isethionate.

The subject compounds can find use in a variety of research applicationsincluding the identification and testing of candidate compounds (e.g.,for pharmaceutical development) and performing research on diseaseconditions of interest in which a target microbe is implicated. Researchapplications of interest can involve use of the subject compounds in avariety of in vitro assays including high throughput screening assays,potency assays, and competitive inhibition assays where the subjectpeptides can be useful as a control compound or as a tool in theinvestigation the sample of interest.

DEFINITIONS

It is to be understood that this invention is not limited to particularembodiments described herein, which as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are described herein.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

The present disclosure provides antimicrobial polypeptides, sometimesreferred to as “RP peptides,” that satisfy one or more of the structuralformulae or sequences described herein. The present disclosure alsoprovides antimicrobial polypeptides that share a minimum degree ofhomology (e.g., as described herein) with any of the exemplary RPpeptides disclosed herein. Thus, a peptide or polypeptide of the presentdisclosure is an antimicrobial polypeptide that satisfies one of theformulae described herein or shares a minimum degree of homology withany of the exemplary RP peptides disclosed herein.

The term “antimicrobial” refers to the ability of a compound, i.e. asubject peptide, to decrease the population of microscopic flora and/orfauna in an environment or sample. Antimicrobial activity includesbacteriostatic or antibacterial activity, antifungal activity, antialgalactivity, and the like. An antimicrobial need not eliminate allmicrobes, but simply decreases the viable population on the treatedsurface. Similarly, “antimicrobial activity” refers to the ability of acompound to inhibit or irreversibly prevent the growth of amicroorganism. Such inhibition or prevention can be through amicrobicidal action or microbistatic inhibition. “Microbicidalinhibition” refers to the ability of the antimicrobial to kill orirrevocably damage the target organism. “Microbistatic inhibition”refers to the ability of the microbistatic or antimicrobial compound toinhibit or to retard the growth of the target organism without causingdeath. Microbicidal or microbistatic inhibition can be applied to citheran environment presently exhibiting microbial growth (i.e., therapeutictreatment) or an environment at risk of supporting such growth (i.e.,prevention or prophylaxis).

“Biofilm” refers any group of microorganisms in which cells stick toeach other on a surface.

The terms “peptide” and “polypeptide” are used interchangeably to referto polymers constructed from amino acid residues.

The term “amino acid residue,” as used herein, refers to any naturallyoccurring amino acid, non-naturally occurring amino acid, or amino acidmimetic (such as a peptoid monomer). An amino acid residue can be in anL- or D-form.

The “length” of a polypeptide is the number of amino acid residueslinked end-to-end that constitutes the polypeptide, excluding anynon-peptide linkers and/or modifications that the polypeptide maycontain.

In certain embodiments of the antimicrobial polypeptides describedherein, a numbering scheme is utilized for convenience and simplicity torefer to particular positions in the structure and/or sequence of thecompounds, e.g., positions at which particular variant amino acidresidues of interest are incorporated into the polypeptide motif. Thisnumbering scheme is based on a sequential order of amino acid residuesin order to assign a numbered location to an amino acid residue ofinterest, e.g., a location in a motif or a structural model as describedherein. By way of example, an antimicrobial peptide of the sequenceFIOKFAKOFKOFIOKFAK (SEQ ID NO: 55), may be described and numberedaccording to the following scheme: F¹I²O³K⁴F⁵A⁶K⁷O⁸F⁹K¹⁰O¹¹F¹²I¹³,O¹⁴K¹⁵F¹⁶A¹⁷K¹⁸ (SEQ ID NO: 55). It is understood that the numbering ofa sequence is not meant to be limiting on the length of a subjectantimicrobial peptide and that one or more additional amino acidresidues and/or terminal modifications may be included at the N-terminaland/or C-terminal of a numbered sequence, b

In certain instances, the subject antimicrobial peptides can includecross-linking residues (e.g., cysteine residues capable of disulfideformation) and/or bubble regions, e.g., a region as described hereinflanked by cysteine residues and/or linking residue(s), such as glycineresidue(s). In such examples, the cross-linking residues are generallynot included in the sequential numbering of the polypeptide motif, butmay include their own separate numbering system. By way of example theantimicrobial peptide of the sequence RFCWKVCYKGICFKKCK (RP557, SEQ IDNO: 8) which includes 4 cysteine residues, may be numbered according tothe following scheme: R¹F²-C¹-W³K⁴V⁵-C²-Y⁶K⁷G⁸I⁹-C³-F¹⁰K¹¹K¹²-C⁴-K¹³(RP557, SEQ ID NO: 8). It is understood that cross-linking residues(e.g., cysteine residues) that are included in a subject peptide maycapable of forming an intramolecular. In some cases, the cross-linkingresidue is a cysteine residue that is capable of forming a disulfidebond with another.

A “linker” or “linker sequence” can be any moiety that links two peptidesequences together. In some embodiments, a linker is an amino acidsequence that is co-linear with the peptide sequences being linkedtogether, whereas in other embodiments a linker is a separate moietythat is attached to the two peptide sequences, e.g., via a covalentlinkage. Linkers can be amino acid sequences or be non-amino acidmoieties. In certain embodiments, a linker is used to facilitatedimerization of two amphipathic regions.

“Pharmaceutically acceptable” as used herein refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals (such as one or more of the animal“patients” or “subjects” as discussed above) without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio. “Pharmaceuticallyacceptable salt”, as used herein, refers to derivatives of the compoundsdefined herein, wherein the parent compound is modified by making acidor base salts thereof.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isaverage molecular weight, temperature is in degrees Centigrade, andpressure is at or near atmospheric.

General Methods of Assessing Peptides Example 1: Activity AgainstPlanktonic Gram-Negative and Gram-Positive Bacteria

Peptides are tested against the following challenge organisms by theM11-A8E CLSI standard for Antimicrobial Susceptibility Testing ofAnaerobic Bacteria: Enterococcus faecium ATCC 700221; Enterobacteraerogens ATCC 13048; Staphylococcus aureus MRSA ATCC 33591;Streptococcus pneumoniae ATCC 49619; Pseudomonas aeruginosa ATCC 27853;Acinetobacter baumannii ATCC 17978D-5; Pseudomonas aeruginosa ATCC19660; and Staphylococcus epidermidis ATCC 51625. Sample dilutions rangefrom initial sample to 1:2048. Eleven (11) concentrations are tested induplicate on a 96 well plate by MQA Laboratories. Results are shown asMinimum bactericidal concentration (MBC), which is the concentration ofeach peptide necessary to yield 99.9% lethality for each of the eightchallenge organisms.

Example 2: Activity Against Biofilm Bacteria

The Minimum Biofilm Eradication Concentration (MBEC) Assay is used. MBECvalues provide estimates on the concentration of an antimicrobialproduct required to kill biofilm bacteria. The Calgary Biofilm Device(CBD) plate is used to effect biofilm formation on a lid containing 96pegs. Bacterial cultures are grown and diluted in Tryptic Soy Broth(TSB) to approximately 1×10⁷ CFU/mL before inoculation of the CBD plate,which are then incubated for 24 hr at 35° C. on a shaker at 125 rpm.

The peg lid containing biofilm is first rinsed in PBS to removeplanktonic cells prior to treatment with 2-fold serial dilutions of testarticles and control overnight at 35° C. The peg lid is rinsed in PBStwice before sonication in fresh media to disrupt biofilm adhered to thepegs. The plate is then incubated overnight to evaluate growth.Bacterial quantification is performed by measuring absorbance at 650 nm(A650). By definition, A650 reading of less than 0.1 indicates biofilmeradication.

Example 3: Activity Against Biothreat Bacteria (B. thailandensis)

In vitro activity of the Test Articles and comparator antibiotic(ceftazidime) are tested as follows: in a sterile 96-well plate, 1×10⁵CFU per well of bacteria are incubated with serial dilutions ofantibiotic (control) and peptide in 10 mM phosphate buffer (3 h, 37°C.). Bacterial survival is determined by serial dilution at each peptideconcentration in sterile PBS. Dilutions are plated in triplicate onnutrient agar and incubated at 37° C. for 24 h; colonies are thencounted to determine survival. Bacterial survival is calculated by theratio of the number of colonies on each experimental plate to theaverage number of colonies in the control plates lacking anyantimicrobial peptide. The antimicrobial peptide concentration requiredto kill 50% of B. thailandensis (EC50) is determined by graphing percentsurvival versus log of peptide concentration. EC50 is determined byfitting the data to a standard sigmoidal dose-response curve. Eachexperiment was performed with three replicates.

Example 4: Antibacterial and Antifungal Activity of Selected Peptides(IC50 Values)

Measurement of antimicrobial and anti-fungal activity is determined by astandard micrometer dilution method. Briefly, cells are grown overnightin media specified for each strain, and are diluted in the same media.Serial dilutions of the peptides are added to microtiter plates in avolume of 50 ul, followed by the addition of 50 ul of bacteria or fungi,5×10⁵ CFU/ml. Plates are incubated at 37 degrees for 24 hours and theMinimum Inhibitory Concentrations (MICs) are determined as the lowestpeptide concentration that inhibited 50% of bacterial growth.

Example 5: Screening of Peptides for In Vitro Bactericidal Activity

Bacteria tested included Burkholderia cepacia strain Toronto (B.c.),Porphyromonas gingivalis strains A7436 and HG405, Actinobacillusactinomycetemcomitans strain A7154 (A.a.). Fusobacterium nucleatumstrain 1594 (F.n.), Escherichia coli strain (E.c.), Staphylococcusaureus ATCC strain 29213 (S.a.), and Pseudomonas aeruginosa strain(P.a.). All bacteria are grown in appropriate media under appropriateatmosphere to the early exponential phase of growth. Media areinoculated with a dose of bacteria to assure a minimum of five doublingsbefore harvesting. The cultures are washed twice in saline bycentrifugation and resuspended in saline at suitable concentration. Inthe initial screening, all peptides are used at a final concentration of10 μM in saline with the target bacteria at 10⁶ CFU/ml as estimated byoptical density at 660 nm. Controls are treated with an equal volume ofsaline. The suspensions are incubated at 37° C. in ambient atmosphereand aliquots removed temporally (0 to 2 hrs) for quantitative recoveryof colony forming units. This allows determination of the kinetics ofkilling of the individual peptides with the different bacterial strains.Killing is considered significant if there is greater than a one-logreduction in recoverable CFU in the peptide-treated vs. thesaline-treated control. Peptides that failed to kill at 10 μM areconsidered inactive. Any peptide that results in greater than two logsreduction is titrated by either two-fold, five-fold or ten-folddilutions prior to testing with 10⁶ CFU/ml of the target bacteria. Theendpoint titration is determined as the last concentration of peptide(in μM) that gives greater than two-logs reduction in recoverable CFUvs. the saline-treated control (“Two-log Reduction Concentration”).

Example 6: Killing of Antibiotic Resistant Bacteria

Staphylococcus aureus, Pseudomonas aeruginosa, and Clostridium difficileare tested for their sensitivity to exemplary subject antimicrobialpolypeptides. These organisms are associated with hospital-acquiredinfections. The experiments are performed as described in Example 5.

Assessment of Activity of Antimicrobial Peptides Example 7: Synthesisand Evaluation of Peptides

Antimicrobial peptides (AMPs) were designed based on sequences found innaturally occurring AMPs. The subject AMPs are amphipathic cationicpeptides with the ability to kill microbes by disrupting their membranefunction. This mode of action rapidly kills antibiotic resistantmicrobes, even in biofilm. In general, the bacteria tested did notdevelop resistance to the subject AMPs.

Thirty-four AMPs were synthesized and evaluated in three iterativerounds. Each AMP was evaluated for antimicrobial potency against 11strains of bacteria, 7 strains of fungi and their cytotoxicitydetermined using L929 fibroblasts and human keratinocytes. The AMPs wereevaluated for biofilm eradication and ability to induce antimicrobialresistance.

From the in vitro screening assays three AMPs were selected for furtherevaluation in a qualified porcine burn wound model infected with P.aeruginosa and S. aureus. Full thickness wounds were created with heatedbrass rod and 2 cm trephine: approximately 10 minutes after homeostasis,wounds were infected with a 2:2:1 mixture of S. aureus, P. aeruginosaand Fuscobacterium ssp. Treatment of infected wounds began 24 hours postinfection to allow for biofilm formation.

Exemplary AMP RP557 is an amphipathic, α-helical molecule having a NP(non-polar hydrophobic face) and a P (polar hydrophilic face). It has 17amino acids and a hairpin structure formed by two disulfide bonds. Thepositive charges on the polar surface are capable of interacting withand disrupting the negatively charged phospholipids on the cellmembranes of microorganisms.

Exemplary AMP RP557 was prepared in one gram lots at 98% purity orbetter. RP557 possesses appropriate physiochemical properties: RP557 ishydrophilic, highly soluble, stable at extremes of pH (<4), resistant toproteases and stable in serum (human serum few 72 hrs at 37° C.monitored via LC-MS/MS). Furthermore, advances in solid and solutionphase peptide chemistry has enabled Good Manufacturing Practice (GMP)material to be produced rapidly, economically in high yield and purity.

Example 8: RPSS7 Activity Results

RP557 possesses broad-spectrum antimicrobial activity against multipleclinical isolates of gram-negative bacteria; A. baumannii and P.aeruginosa; gram-positive bacteria, S. aureus and MRSA and fungalstrains; C. albicans, C. parapsilosis, C. krusei, Aspergillus fumigatus,Aspergillus flavus, Absidia corymbifera, Fusarium solani and Mucor.

In vitro time-kill assays demonstrated RP557 quickly kills bothGram-positive and Gram-negative bacteria, at low doses (1 to 2 μg/mL)and are not cytotoxic to mammalian cells (FIG. 3). The rapid destructionof pathogenic cells by potent broad-spectrum topical anti-infectivesinfer a theoretical reduced likelihood of developing bacterialresistance. FIG. 3A to 3C shows RP557 rapidly eradicates P. aeruginosaand S. aureus with no cytotoxicity to mammalian cells. Cell viabilitywas performed using bioluminescent strains of P. aeruginosa 19660 (FIG.3A), S. aureus 49525 (FIG. 3B) & L929 fibroblast cells (FIG. 3C) andimaged with the I VIS Lumina system. Data represents the mean of 3measurements.

Pathogens do not Develop Resistance Against RP557

Neither P. aeruginosa nor S. aureus bacteria become resistant tosub-inhibitory concentrations of RP557 after 30 rounds of selectionwhereas for the standard antibiotics resistance did develop, asevidenced by gentamicin and clindamycin showing growth at 4096 and 256times the minimum inhibitory concentration (MIC) after 30 days againstP. aeruginosa and S. aureus, respectively (FIG. 4A to 4B). The resistantstrains at the end of 30 serial passages were then treated with RP557and both strains were found to be readily susceptible therefore, thereis no cross resistance to RP557 and gentamicin-resistant P. aeruginosaand clindamycin-resistant S. aureus.

FIG. 4A to 4B. P. aeruginosa and S. aureus did not develop resistanceagainst RP557. Sub-inhibitory concentrations of RP557, gentamicin andclindamycin were incubated with P. aeruginosa 27853 and S. aureus 29213for 24 hours. Bacteria showing growth in the highest concentration werere-passaged in fresh dilutions containing sub-minimum inhibitoryconcentration (MIC) levels of each component for 30 consecutivepassages; means are shown.

Biofilm Inactivation

RP557 prevents and treats biofilms of various bacterial and fungalspecies found in combat wounds thereby mitigating the influence ofinfection on healing and regeneration. A representative study of RP557against Candida albicans, is shown in FIG. 5A to 5B.

FIG. 5A to 5B: RP557 is a potent inhibitor of Candida biofilm.Fluconazole or RP557 were added to preformed Candida 17-88 biofilm for24 hours and biofilm inhibition evaluated via metabolic evaluation usingXTT, 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)[phenyl-amino)carbonyl]-2H. Data represent the mean±SD of triplicatemeasurements and statistical significance, compared to vehicle control,determined by one-way ANOVA followed by Dunnett's test (*p<0.05,**p<0.01, and ***p<0.001). These results indicate the biofilm is notresistant to antimicrobial peptides such as RP557.

Bactericidal In Vivo Activity

RP557 dose-dependently reduced both P. aeruginosa and S. aureus in apolymicrobial infected full-thickness thermal porcine burn model (FIG.6A-6B). A single topical application of RP557 reduced S. aureus CFUcounts from vehicle control levels of 4.81±0.724 log CFU/g tissue to4.20±1.19, 2.11±1.75 and 1.46±2.05 log CFU/g tissue for 0.1%, 0.2%(p<0.01) and 1% RP557 (p<0.0001), respectively. Furthermore, a dose of2% RP557 completely eradicated the S. aureus infection. Moreover, asingle RP557 dose reduced the number of viable P. aeruginosa bacteriafrom 5.87±0.477 log CFU/g tissue to 2.34±2.10 and 1.96±1.64 log CFU/gtissue respectively for 1% (p<0.0001) and 2% (p<0.0001), respectively.

FIG. 6A to 6B shows that topical treatment with exemplary peptide RP557reduces polymicrobial infection with dose response in an infectedporcine burn model. Full thickness wounds were created with a heatedbrass rod and a trephine on the back of anesthetized pigs followed byinfection with 2:2:1 mixture of S. aureus ATCC 6538 (FIG. 6B) and P.aeruginosa (porcine isolate) (FIG. 6A) and fusobacterium ssp. After 3hours, 0, 0.1, 0.2, 1 or 2% RP557 were applied. After 24 hours, woundswere sampled by punch biopsy and bacterial counts, expressed as log(Colony Forming Units, CFUs/g) evaluated. Data is expressed as mean±SEof 8 replicates. Statistical significance, compared to vehicle,determined by one-way ANOVA followed by Dunnett's test (**p<0.01,****p<0.0001).

RP557 Kills Infection Throughout a 24 Hour Period.

FIG. 7A-7B Pharmacodynamic Response to RP557. Twenty four hours postbacterial inoculation biofilm associated wounds were treated with 2%RP557. Punch biopsies were taken at 30 min, 180 min and 24 hours posttreatment, homogenized, plated and CFUs counted. Statisticalsignificance was calculated using the Holm-Sidak method. *p<0.05,***p<0.001, ****p<0.0001, dAMP vs Control.

Fungicidal In Vivo Activity

RP557 was effective in eradicating C. albicans in a rodent model ofvulvovaginal candidiasis (FIG. 8A-8B). RP557 resulted in significantreductions in fungal counts relative to the vehicle control group(****p<0.0001). Furthermore, the results were more effective than oralfluconazole which exhibited minimal activity.

FIG. 8A to 8B. RP557 topical treatment reduces rodent vaginalcandidiasis. Effects of RP557, miconazole and oral fluconazole in the C.albicans (ATCC 44858) vaginal infection rodent model. On Day 0, ratswere inoculated intravaginally (IVG) with C. albicans at 1.46×107CFU/rat. RP557 and miconazole at 20 mg/mL were administered IVG at 0.1mL/rat twice daily at 8 hr intervals starting from 48 hr after infectionfor 3 days. CPUs were evaluated on Day 5 with limit of detection (LOD)0.7 CFU/rat. Significant difference, compared to vehicle, determined byone-way ANOVA followed by Dunnett's test (*p<0.05, ****p<0.0001). Aseparate group receiving oral fluconazole; *p<0.05 versus 0.1 and 10mg/kg fluconazole.

The results provided in the Examples demonstrate the efficacy of theantimicrobial peptides of the invention in killing a wide range ofmicrobial organisms, including those that cause medically importanthuman infections.

CLAUSES

Notwithstanding the appended claims, the following clauses are providedto illustrate aspects of the present disclosure.

Clause 1. An antimicrobial peptide, comprising:

-   -   a) a peptide sequence selected from RP550-567 (SEQ ID NO: 1 to        SEQ ID NO 18);    -   b) a sequence having at least 80% sequence identity (e.g., at        least 85%, at least 90%, or at least 95% sequence identity) with        the sequence defined in a); or    -   c) a sequence having five or less (e.g., four or less, three or        less, two or less such as one or two)amino acid substitutions        relative to the sequence defined in a), wherein the five or less        (e.g., four or less, three or less, two or less such as one or        two) amino acid substitutions are substitutions for amino acids        according to Table 2 (e.g., a similar amino acid substitution, a        conservative amino acid substitution or a highly conservative        amino acid substitution).

Clause 2. The antimicrobial peptide of clause 1, comprising:

-   -   a) a peptide sequence selected from RP550-567 (SEQ ID NO: 1 to        SEQ ID NO 18); or    -   b) a sequence having five or less (e.g., four or less, three or        less, two or less such as one or two)amino acid substitutions        relative to the sequence defined in a), wherein the five or less        (e.g., four or less, three or less, two or less such as one or        two)amino acid substitutions consist of substitution of a        cationic amino acid of the sequence with an alternative cationic        amino acid residue (e.g., K for O, O for K, K for R, etc.).

Clause 3. The antimicrobial peptide of clause 1, comprising the peptidesequence selected from RP550-567 (SEQ ID NO: 1 to SEQ ID NO 18).

Clause 4. The antimicrobial peptide of clause 1, consisting of thepeptide sequence selected from RP550-567 (SEQ ID NO: 1 to SEQ ID NO 18).

Clause 5. The antimicrobial peptide of clause 1, comprising the peptidesequence of formula 7A¹:

(7A¹) (SEQ ID NO: 47) X¹J²-C¹-J³X⁴V-C²-YX⁷GI-C³-J¹⁰X¹¹X¹²-C⁴-X¹³

-   -   Wherein:    -   X¹ is selected from O and R;    -   J² and J³ are each independently selected from F and W;    -   X⁴, X⁷, X¹¹, X¹² and X¹³ are each independently selected from O        and K; and    -   J¹⁰ is selected from Y and F.

Clause 6. The antimicrobial peptide of clause S, wherein the peptidesequence of formula 7A¹ is selected from the group consisting of:RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8), RWCFKVCYKGICYKKCK (RP560) (SEQID NO: 11), OWCFOVCYOGICYOOCO (RP559) (SEQ ID NO: 10), OFCWOVCYOGICFOOCO(RP561) (SEQ ID NO: 12).

Clause 7. The antimicrobial peptide of clause 1, comprising the peptidesequence of formula 7B¹:

(7B¹) (SEQ ID NO: 49) X¹-C¹-X²X³J⁴-C²-IGX⁷Y-C³-VX¹⁰J¹¹-C⁴-J¹²X¹³

-   -   wherein X¹, X², X³, X⁷ and X¹⁰ are each independently selected        from O and R;    -   J⁴ is selected from Y and F;    -   J¹¹ and J¹² are each independently selected from F and W; and    -   X¹³ is selected from K and O.

Clause 8. The antimicrobial peptide of clause 7, wherein the peptidesequence of formula 7B¹ is selected from the group consisting of:RCRRYCIGRYCVRFCWK (RP558) (SEQ ID NO: 9) and OCOOFCIGOYCVOWCFO (RP562)(SEQ ID NO: 13).

Clause 9. The antimicrobial peptide of clause 1, comprising the peptidesequence of formula 6A:

(6A) (SEQ ID NO: 27) FX²IX⁴AX⁶L(X⁸)_(m)-B-(X¹³)_(m)-IX¹⁵AX¹⁷LX¹⁹F

-   -   wherein:        -   B is a sequence selected from CLGX¹¹FC (SEQ ID NO: 28),            GCLGX¹¹FCG (SEQ ID NO: 29) and GGCLGX^(n)FCGG (SEQ ID NO:            30), wherein each C is a cysteine residue, each G is a            glycine residue and X¹¹ is selected from O and K;        -   X², X⁴, X⁸, X¹³, X¹⁵ and X¹⁹ are each independently selected            from O and K;        -   X⁶ and X¹⁷ are each independently selected from R and O; and        -   m is an integer selected from 0 or 1.

Clause 10. The antimicrobial peptide of clause 9, wherein the peptidesequence of formula 6A is selected from the group consisting of:FKIOARLCLGOFCIOARLK (RP550) (SEQ ID NO: 1), FOIOAOLGGCLGOFCGGIOAOLOF(RP564) (SEQ ID NO: 15), FOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO:16), FOIKAOLGGCLGKFCGGIKAOLKF (RP566) (SEQ ID NO: 17) andFOIKAOLKGGCLGKFCGGKIKAOLKF (RP 567) (SEQ ID NO: 18).

Clause 11. The antimicrobial peptide of clause 1, comprising the peptidesequence of formula 3A:

(3A) FX²J³X⁴J⁵X⁶J⁷X⁸J⁹X¹⁰J¹¹X¹²J¹³X¹⁴J¹⁵X¹⁶J¹⁷ 

-   -   wherein:    -   X² and X¹⁴ are each independently selected from O and R;    -   J³ and J¹⁷ are each independently selected from L and I;    -   X⁴ and X¹⁶ are each independently selected from K and O;    -   J⁵ is selected from A and I;    -   X⁶, X⁸, X¹⁰ and X¹² are each independently selected from R, K        and O;    -   J⁷ is selected from F, A and I;    -   J⁹ is selected from V and L;    -   J¹¹ is selected from A, V and L;    -   J¹³ is selected from A, I and L; and    -   J¹⁵ is selected from I, F and L.

Clause 12. The antimicrobial peptide of clause 11, wherein the peptidesequence of formula 3A is selected from the group consisting of:FOIKARFOVRARLOLKI (RP553) (SEQ ID NO: 4), FOLOAOIOVOLOAOIOL (RP555) (SEQID NO: 6), FOLOAOIKVKLOAOIOL (RP556) (SEQ ID NO: 7) andFRLKIKARLKVKIRFKL (RP554) (SEQ ID NO: 5).

Clause 13. The antimicrobial peptide of any one of clauses 1-4, whereinthe peptide sequence selected from RP550-567 is RFCWKVCYKGICFKKCK(RP557) (SEQ ID NO: 8).

Clause 14. The antimicrobial peptide any one of clauses 1-4, wherein thepeptide sequence selected from RP550-567 is FKIOARLCLGOFCIOARLK (RP550)(SEQ ID NO: 1).

Clause 15. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is FIOKFAKOFKOFIOKFAKFAFAF(RP551) (SEQ ID NO: 2).

Clause 16. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is FAFAFKAFKKAFKOFOOAFOOAF(RP552) (SEQ ID NO: 3).

Clause 17. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is FOIKARFOVRARLOLKI(RP553) (SEQ ID NO: 4).

Clause 18. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is FRLKIKARLKVKIRFKL(RP554) (SEQ ID NO: 5).

Clause 19. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is FOLOAOIOVOLOAOIOL(RP555) (SEQ ID NO: 6).

Clause 20. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-557 is FOLOAOIKVKLOAOIOL(RP556) (SEQ ID NO: 7).

Clause 21. The antimicrobial peptide any one of clauses 1-4, wherein thepeptide sequence selected from RP550-567 is RCRRYCIGRYCVRFCWK (RP558)(SEQ ID NO: 9).

Clause 22. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is OWCFOVCYOGICYOOCO(RP559) (SEQ ID NO: 10).

Clause 23. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is RWCFKVCYKGICYKKCK(RP560) (SEQ ID NO: 11).

Clause 24. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is OFCWOVCYOGICFOOCO(RP561) (SEQ ID NO: 12).

Clause 25. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is OCOOFCIGOYCVOWCFO(RP562) (SEQ ID NO: 13).

Clause 26. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-567 is RGVCVCFRRRCYCLRGGR(RP563) (SEQ ID NO: 14).

Clause 27. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-557 is FOIOAOLGGCLGOFCGGIOAOLOF(RP564) (SEQ ID NO: 15).

Clause 28. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-557 isFOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO: 16).

Clause 29. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-557 is FOIKAOLGGCLGKFCGGIKAOLKF(RP566) (SEQ ID NO: 17).

Clause 30. The antimicrobial peptide of any one of claims 1-4, whereinthe peptide sequence selected from RP550-557 isFOIKAOLKGGCLGKFCGGKIKAOLKF (RP567) (SEQ ID NO: 18).

Clause 31. An antimicrobial peptide, comprising the peptide sequence offormula (7A¹):

(7A¹) (SEQ ID NO: 47) X¹J²-C¹-J³X⁴V-C²-YX⁷GI-C³-J¹⁰X¹¹X¹²-C⁴-X¹³

-   -   Wherein:    -   X¹ is selected from O and R;    -   J² and J³ are each independently selected from F and W;    -   X⁴, X⁷, X¹¹, X¹² and X¹³ are each independently selected from O        and K; and    -   J¹⁰ is selected from Y and F.

Clause 32. The antimicrobial peptide of clause 31, wherein the peptidesequence of formula 7A¹ is selected from the group consisting of:RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8), RWCFKVCYKGICYKKCK (RP560) (SEQID NO: 11), OWCFOVCYOGICYOOCO (RP559) (SEQ ID NO: 10), OFCWOVCYOGICFOOCO(RP661) (SEQ ID NO: 12).

Clause 33. An antimicrobial peptide, comprising the peptide sequence offormula (7B¹)

(7B¹) (SEQ ID NO: 49) X¹-C¹-X²X³J⁴-C²-IGX⁷Y-C³-VX¹⁰J¹¹-C⁴-J¹²X¹³

-   -   wherein X¹, X², X³, X⁷ and X¹⁰ are each independently selected        from O and R;    -   J⁴ is selected from Y and F;    -   J¹¹ and J¹² are each independently selected from F and W; and    -   X¹³ is selected from K and O.

Clause 34. The antimicrobial peptide of clause 33, wherein the peptidesequence of formula 7B′ is selected from the group consisting of:RCRRYCIGRYCVRFCWK (RP558) (SEQ ID NO: 9), OCOOFCIGOYCVOWCFO (RP562) (SEQID NO: 13).

Clause 35. An antimicrobial peptide, comprising the peptide sequence offormula (6A)

(6A) (SEQ ID NO: 27) FX²IX⁴AX⁶L(X⁸)_(m)-B-(X¹³)_(m)-IX¹⁵AX¹⁷LX¹⁹F

-   -   wherein:    -   B is a sequence selected from CLGX¹¹FC (SEQ ID NO: 28),        GCLGX¹¹FCG (SEQ ID NO: 29) and GGCLGX^(n)FCGG (SEQ ID NO: 30),        wherein each C is a cysteine residue, each G is a glycine        residue and X¹¹ is selected from O and K; X², X⁴, X⁸, X¹³, X¹⁵        and X¹⁹ are each independently selected from O and K;    -   X⁶ and X¹⁷ are each independently selected from R and O; and    -   m is an integer selected from 0 or 1.

Clause 36. The antimicrobial peptide of clause 35, wherein the peptidesequence of formula 6A is selected from the group consisting of:FKIOARLCLGOFCIOARLK (RP550) (SEQ ID NO: 1), FOIOAOLGGCLGOFCGGIOAOLOF(RP564) (SEQ ID NO: 15), FOIOAOLOGGCLGOFCGGOIOAOLOF (RP565) (SEQ ID NO:16), FOIKAOLGGCLGKFCGGIKAOLKF (RP566) (SEQ ID NO: 17),FOIKAOLKGGCLGKFCGGKIKAOLKF (RP 567) (SEQ ID NO: 18).

Clause 37. An antimicrobial peptide, comprising the peptide sequence offormula (3A)

(3A) FX²J³X⁴J⁵X⁶J⁷X⁸J⁹X¹⁰J¹¹X¹²J¹³X¹⁴J¹⁵X¹⁶J¹⁷ 

-   -   wherein:    -   X² and X¹⁴ are each independently selected from O and R;    -   J³ and J¹⁷ are each independently selected from L and I;    -   X⁴ and X¹⁶ are each independently selected from K and O;    -   J⁵ is selected from A and I;    -   X⁶, X⁸, X¹⁰ and X¹² are each independently selected from R, K        and O;    -   J⁷ is selected from F, A and I;    -   J⁹ is selected from V and L;    -   J¹¹ is selected from A, V and L;    -   J¹³ is selected from A, I and L; and    -   J¹⁵ is selected from I, F and L.

Clause 38. The antimicrobial peptide of clause 37, wherein the peptidesequence of formula 3A is selected from the group consisting of:FOIKARFOVRARLOLKI (RP553) (SEQ ID NO: 4), FOLOAOIOVOLOAOIOL (RP555) (SEQID NO: 6). FOLOAOIKVKLOAOIOL (RP556) (SEQ ID NO: 7), FRLKIKARLKVKIRFKL(RP554) (SEQ ID NO: 5).

Clause 39. A pharmaceutical composition, comprising the antimicrobialpeptide of any one of clauses 1-38 and a pharmaceutically acceptablecarrier.

Clause 40. The pharmaceutical composition of clause 39, wherein thecomposition is formulated for oral administration, parenteraladministration, or topical administration.

Clause 41. The pharmaceutical composition of clause 39, wherein thecomposition is formulated for oral administration and further comprisesan enteric coating.

Clause 42. The pharmaceutical composition of clause 39, wherein thecomposition is formulated for topical delivery in a form selected fromthe group consisting of: a gel suspension, a cream, microneedle, andinfused into a bandage or topical patch.

Clause 43. The pharmaceutical composition of clause 39, wherein thecomposition is formulated for inhalation.

Clause 44. The pharmaceutical composition of clause 39, wherein thecomposition is an ophthalmic composition formulated for delivery to theeye of a subject, e.g., via topical instillation, subconjunctival,subtenon, intravitreal, retrobulbar, or intracameral administration.

Clause 45. The pharmaceutical composition of clause 43, wherein thecomposition further comprises liposomes comprising free and/orencapsulated antimicrobial peptide.

Clause 46. The pharmaceutical composition of any one of clauses 39-45,further comprising an additional bioactive agent.

Clause 47. The pharmaceutical composition of clause 46, wherein theadditional bioactive agent is selected from an antimicrobial agent, ananti-inflammatory drug, an anti-nausea drug, an anti-pain medication,and combinations thereof.

Clause 48. The pharmaceutical composition of clause 39, wherein thecomposition is formulated to be coated on the surface of an implantablemedical device.

Clause 49. The pharmaceutical composition of clause 48, wherein themedical device is selected from surgical instruments and indwellingmedical devices.

Clause 50. A method of treating or preventing a microbial infection(e.g., as described herein) in a subject in need thereof, the methodcomprising administering a pharmaceutical composition according to anyone of clauses 39-49 to the subject.

Clause 51. The method of clause 50, wherein the pharmaceuticalcomposition is administered to the subject orally, parenterally, viainhalation or topically.

Clause 52. The method of clause 50, wherein the pharmaceuticalcomposition is an ophthalmic composition administered to the eye of asubject (e.g., as described herein via topical instillation,subconjunctival, subtenon, intravitreal, retrobulbar, or intracameralroute of administration).

Clause 53. The method of clause 50, wherein the pharmaceuticalcomposition is administered to the subject by applying the compositionto a surface of a medical device prior to inserting the medical deviceinto the subject.

Clause 54. The method of any one of clauses 50-53, wherein the subjectis selected from the group consisting of: a human, a domesticatedanimal, a farm animal, and a zoo animal.

Clause 55. The method of any one of clauses 50-54, wherein thepharmaceutical composition is administered in combination with anantimicrobial agent and/or antibiotic.

Clause 56 A method of treating microbial infection in an animal,comprising administering to an infected or at-risk animal apharmaceutical or veterinary product, a medical device or a dietaryproduct comprising a peptide (e.g., as described herein) in an amounteffective to enhance growth and weight gain of the animal.

Clause 57. A method of inhibiting a microbial growth, colony orinfection (e.g., as described herein) in a sample comprising microbes,the method comprising contacting the sample with an antimicrobialpeptide according to any one of clauses 1-38 to inhibit the microbialgrowth, colony or infection.

Clause 58. The method of clause 57, wherein the sample is a cell sample.

It will also be recognized by those skilled in the art that, while theinvention has been described above in terms of preferred embodiments, itis not limited thereto. Various features and aspects of the abovedescribed invention may be used individually or jointly. Further,although the invention has been described in the context of itsimplementation in a particular environment, and for particularapplications those skilled in the art will recognize that its usefulnessis not limited thereto and that the present invention can bebeneficially utilized in any number of environments and implementations.Accordingly, the claims set forth below should be construed in view ofthe full breadth and spirit of the invention as disclosed herein.

What is claimed:
 1. A peptide, comprising: a) a peptide sequence selected from RP550-557 (SEQ ID NO: 1 to SEQ ID NO: 8), RP559-562 (SEQ ID NO: 10 to SEQ ID NO: 13), RP566 (SEQ ID NO: 17) or RP567 (SEQ ID NO: 18); b) a sequence having at least 90% sequence identity with the sequence defined in a); or c) a sequence having three or less amino acid substitutions relative to the sequence defined in a), wherein the three or less amino acid substitutions are conservative amino acid substitutions according to Table
 3. 2. The peptide of claim 1, comprising: a) a peptide sequence selected from RP550-557 (SEQ ID NO: 1 to SEQ ID NO: 8), RP559-562 (SEQ ID NO: 10 to SEQ ID NO: 13), RP566 (SEQ ID NO: 17) or RP567 (SEQ ID NO: 18); or b) a sequence having three or less amino acid substitutions relative to the sequence defined in a), wherein the three or less amino acid substitutions consist of substitution of a cationic amino acid of the sequence with an alternative cationic amino acid residue.
 3. The peptide of claim 1, wherein the peptide sequence defined in a) is RFCWKVCYKGICFKKCK (RP557) (SEQ ID NO: 8).
 4. The peptide of claim 1, wherein the peptide sequence defined in a) is FOLOAOIOVOLOAOIOL (RP555) (SEQ ID NO: 6).
 5. The peptide of claim 1, wherein the peptide sequence defined in a) is RWCFKVCYKGICYKKCK (RP560) (SEQ ID NO: 11).
 6. The peptide of claim 1, wherein the peptide sequence defined in a) is FKIOARLCLGOFCIOARLK (RP550) (SEQ ID NO: 1).
 7. The peptide of claim 1, wherein the peptide sequence defined in a) is FIOKFAKOFKOFIOKFAKFAFAF (RP551) (SEQ ID NO: 2).
 8. The peptide of claim 1, wherein the peptide sequence defined in a) is FAFAFKAFKKAFKOFOOAFOOAF (RP552) (SEQ ID NO: 3).
 9. The peptide of claim 1, wherein the peptide sequence defined in a) is FOIKARFOVRARLOLKI (RP553) (SEQ ID NO: 4).
 10. The peptide of claim 1, wherein the peptide sequence defined in a) is FOLOAOIKVKLOAOIOL (RP556) (SEQ ID NO: 7).
 11. The antimicrobial peptide of claim 1, comprising a peptide sequence having at least 90% sequence identity with a sequence selected from RP550-557 (SEQ ID NO: 1 to SEQ ID NO: 8), RP559-562 (SEQ ID NO: 10 to SEQ ID NO: 13), RP566 (SEQ ID NO: 17) or RP567 (SEQ ID NO: 18).
 12. The peptide of claim 1, comprising a peptide sequence selected from: OFCWOVCYOGICFOOCO (RP561) (SEQ ID NO: 12); and OWCFOVCYOGICYOOCO (RP559) (SEQ ID NO: 10).
 13. A pharmaceutical composition, comprising the peptide of claim 1 and a pharmaceutically acceptable carrier.
 14. The pharmaceutical composition of claim 13, wherein the composition is formulated for oral administration, parenteral administration, inhalation administration or topical administration.
 15. The pharmaceutical composition of claim 13, wherein the composition is formulated for oral administration and further comprises an enteric coating.
 16. The pharmaceutical composition of claim 13, wherein the composition is formulated for topical delivery in a form selected from a gel suspension, a cream, microneedle, and infused into a bandage or topical patch.
 17. The pharmaceutical composition of claim 13, wherein the composition is formulated for inhalation.
 18. The pharmaceutical composition of claim 13, further comprising an additional bioactive agent.
 19. The pharmaceutical composition of claim 18, wherein the additional bioactive agent is selected from an antimicrobial agent, an anti-inflammatory drug, an anti-nausea drug, an anti-pain medication, and combinations thereof.
 20. The pharmaceutical composition of claim 13, wherein the composition is formulated to be coated on the surface of an implantable medical device.
 21. A method of reducing the likelihood of a microbial infection in a subject in need thereof, the method comprising administering a pharmaceutical composition according to claim 13 to the subject.
 22. The method of claim 21, wherein the pharmaceutical composition is administered to the subject orally, parenterally, via inhalation or topically.
 23. The method of claim 21, wherein the pharmaceutical composition is administered to the subject by applying the composition to a surface of a medical device prior to inserting the medical device into the subject.
 24. The method of claim 21, wherein the subject is selected from a human, a domesticated animal, a farm animal and a zoo animal. 